Cloning and recombinant production of CRF receptor(s)

ABSTRACT

In accordance with the present invention, there are provided novel G-protein-coupled receptor proteins (CRF-R) characterized by having sufficient binding affinity for corticotropin releasing factor (CRF) such that concentrations of £ 10 nM of CRF occupy  3 50% of the binding sites of said receptor protein. Nucleic acid sequences encoding such receptors, assays employing same, as well as antibodies derived therefrom, are also disclosed. Invention CRF-Rs can be employed in a variety of ways, such as, for example, in bioassays, for production of antibodies thereto, in therapeutic compositions containing such proteins and/or antibodies.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/483,139,filed Jun. 7, 1995, which is a continuation-in-part of U.S. Ser. No.08/353,537, filed Dec. 9, 1994, now pending, which is acontinuation-in-part of PCT Application No. PCT/US94/05908, filed May25, 1994, now pending, which is a continuation-in-part of U.S. Ser. No.08/110,286, filed Aug. 23, 1993, now pending, which is acontinuation-in-part of U.S. Ser. No. 08/079,320, filed Jun. 18, 1993,now abandoned.

ACKNOWLEDGEMENT

This invention was made with United States Government support underGrant Number DK26745, awarded by the National Institutes of Health. TheUnited States Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to receptor proteins, DNA sequencesencoding same, and various uses therefor.

BACKGROUND OF THE INVENTION

Corticotropin-releasing factor (CRF) is a 41-residue hypothalamicpeptide which stimulates the secretion and biosynthesis of pituitaryadrenocorticotrophic hormone (ACTH) leading to increased adrenalglucocorticoid production. CRF was originally isolated and characterizedon the basis of its role in this hypothalamic-pituitary-adrenal axis(HPA) [Vale et al., Science Vol. 213:1394-1397 (1981)]. More recently,however, CRF has been found to be distributed broadly within the centralnervous system. (CNS) as well as in extra-neural tissues such as theadrenal glands and testes [Swanson et al., Neuroendocrinology Vol.36:165-186 (1983); Suda et al., J. Clin. Endocrinol. Metab. Vol.58:919-924 (1984; Fabbri and Dufau, Endocrinology Vol. 127:1541-1543(1990)], and sites of inflammation, where it may also act as a paracrineregulator or neurotransmitter.

In addition to the critical role of CRF in mediating HPA axisactivation, it has been shown to modulate autonomic and behavioralchanges that occur during the stress response. Many of these behavioralchanges have been shown to occur independently of HPA activation in thatthey are insensitive to dexamethasone treatment and hypophysectomy[Britton et al., Life Sci. Vol. 38:211-216 (1986); Britton et al., LifeSci. Vol. 39:1281-1286 (1986); Berridge and Dunn, Pharm. Bioch. Behav.Vol. 34:517-519 (1989)]. In addition, direct infusion of CRF into theCNS mimics autonomic and behavioral responses to a variety of stressors[Sutton et al., Nature Vol. 297:331-333 (1982); Brown and Fisher, BrainRes. Vol. 280:75-79 (1983); Stephens et al., Peptides Vol. 9:1067-1070(1988); Butler et al., J. Neurosci. Vol. 10:176-183 (1990)].Furthermore, peripheral administration of CRF or the CRF antagonist,a-helical CRF 9-41, failed to affect these changes, thus supporting adirect brain action for CRF in such functions. CRF antagonists givenperipherally attenuate stress-mediated increases in ACTH secretion, andwhen delivered into the cerebral ventricles can mitigate stress inducedchanges in autonomic activity and behavior.

As a result of the extensive anatomical distribution and multiplebiological actions of CRF, this regulatory peptide is believed to beinvolved in the regulation of numerous biological processes. The peptidehas been implicated in the regulation of inflammatory responses. On theone hand, it has been observed that CRF plays a pro-inflammatory role incertain animal models, while in others CRF can suppress inflammation byreducing injury induced increases in vascular permeability.

It has also been found that CRF can modify steroid production by thegonads, placenta, and adrenal glands. CRF also has vascular effects suchas dilating the superior mesenteric arterial bed and dilating thecoronary arteries. In addition to CRF acting on the central nervoussystem to modify gastrointestinal function, CRF has been found todirectly effect the gastrointestinal tract as well.

In order to more fully investigate the role of CRF within the endocrine,gastrointestinal, reproductive, central nervous and immune systems, andthe possible interactions of CRF with its cognate receptor, it would bedesirable to have available a ready source of CRF receptor. Furthermore,the availability of recombinant receptor would allow the development ofless expensive, more sensitive, and automated means for assaying CRF andCRF-like compounds and developing CRF-based therapeutics.

The responsivity to CRF or the quantity of CRF receptors in targettissues has been shown or predicted (from altered sensitivity to CRF) tochange in response to a variety of circumstances including Alzheimer'sDisease, melancholic depression, anorexia nervosa, Cushing's Disease,alcoholism, and the like. Thus, the development of specific anti-CRF-Rantibodies and molecular probes for CRF receptor(s) are desired for usein appropriate diagnostic assays.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided newG-protein-coupled receptor proteins which have high binding affinity forcorticotropin-releasing factor (CRF), said proteins are referred tohereinafter as CRF-receptor(s) (CRF-Rs). Invention receptor(s) areprincipal neuroregulators of the hypothalamic-pituitary-adrenal corticalaxis and play an important role in coordinating the endocrine, autonomicand behavioral responses to stress and immune challenge. CRF-Rs arefunctionally coupled to adenylate cyclase as it transduces the signalfor CRF-stimulated intracellular cAMP accumulation. Invention CRF-Rs canbe employed in a variety of ways, such as, for example, in bioassays,for production of antibodies thereto, in therapeutic compositionscontaining such proteins and/or antibodies, and the like.

In accordance with another aspect of the present invention, bindingassays employing CRF-Rs are provided, useful for rapidly screening alarge number of compounds to determine which compounds (e.g., agonistsand antagonists) are capable of binding to the receptors of theinvention. The invention binding assays may also be employed to identifynew CRF-like ligands (e.g., putative mammalian sauvagine or urotensin).Test samples (e.g., biological fluids) may also be subjected toinvention binding assays to detect the presence or absence of CRF orCRF-like compounds.

In accordance with the present invention, recombinant DNA moleculesencoding CRF-Rs are also provided. DNA molecules encoding CRF-Rs (orfragments thereof) are useful, for example, as probes for detecting thepresence of CRF-R encoding nucleic acids in biological samples, theidentification of additional CRF receptor proteins, as coding sequenceswhich can be used for the recombinant expression of the inventionreceptor proteins (or functional fragments thereof), and the like.Recombinant human CRF-Rs have been expressed in COS cells and bind toCRF and CRF analogs with high affinity. The recombinant production ofCRF-Rs makes feasible their use in the foregoing manners. Fragments ofCRF-R encoding nucleic acid can also be employed as primers for PCRamplification of CRF-R encoding DNA. In addition, sequences derived fromsequences encoding CRF-Rs can also be used in gene therapy applicationsto target the expression of vectors carrying useful genes to specificcell types.

In accordance with another aspect of the present invention, anti-CRF-Rantibodies are also provided. CRF-R and anti-CRF-R antibodies are usefulfor diagnostic assays to determine levels of CRF-Rs in various tissuesamples, e.g., neoplastic tissues, and the like. Anti-CRF-R antibodiescan also be used to purify CRF-R protein. Moreover, these antibodies areconsidered therapeutically useful to counteract or supplement thebiological effect of CRF-Rs in vivo.

Methods and diagnostic systems for determining the levels of CRF-R invarious tissue samples, and levels of CRF-R peptide fragments and CRF invascular fluid samples, are also provided. These diagnostic methods canbe used, for example, for monitoring the level of therapeuticallyadministered CRF-R (or fragments thereof) to facilitate the maintenanceof therapeutically effective amounts. These diagnostic methods can alsobe used to diagnose physiological disorders that result from abnormallevels of CRF or CRF-R.

CRF-Rs, fragments thereof that bind CRF, or analogs thereof, are capableof therapeutically modulating the effect of CRF. For example, CRF-Rfragments can inhibit CRF binding to CRF-R and can inhibit CRF-inducedACTH release in vitro by pituitary cells. Thus, CRF-Rs can beadministered therapeutically in mammals to reduce high ACTH levelscaused by excess CRF. Such treatments can be used, for example, to treatCushing's Disease, and the like. These CRF-Rs are also useful incombating pituitary tumors that produce CRF. Moreover, they can be usedto reduce pituitary ACTH secretion and hence reduce cortisol levelsunder any condition in which they are abnormally high, such as, forexample, during chronic stress, in patients afflicted with anorexianervosa or alcoholism, and the like. CRF-Rs administered intravenously(IV) are effective to prevent CRF-induced ACTH release. Furthermore, itis contemplated that IV administration of CRF-Rs can reduce intestinaltransit time and thus combat irritable bowel syndrome.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the pharmacologic characteristics of plasmid hctCRFR(“human Cushing's Tumor Corticotropin-releasing factor-receptor”;encoding CRF receptor subtype hCRF-RA₁), transiently expressed in COSM6cells. FIG. 1 presents the results of displacement of ¹²⁵I(Nle²¹, Tyr³²)ovine CRF (oCRF) by r/hCRF, when oCRF is bound to membranes preparedfrom COSM6 cells transfected with hctCRF receptor (n), or rGnRHR (″), asdescribed in Example 3. The data are from one representative experimentrepeated at least four times.

FIG. 2A illustrates the stimulation of intracellular cAMP in COSM6 cells(transfected with plasmid hctCRF, which encodes CRF receptor subtypeCRF-RA₁) by exposure to CRF, hGRF(1-40)OH, VIP, and Salmon Calcitonin,as described in Example 4.

FIG. 2B illustrates the dose-response stimulation of cAMP in COSM6 cells(transfected with plasmid hctCRFR, which encodes CRF receptor subtypeCRF-RA₁) by increasing concentrations of CRF in cells pretreated (n) oruntreated (″) with the phosphodiesterase inhibitor, IBMX(3-isobutyl-1-methylxanthine).

FIG. 2C illustrates the inhibition of CRF stimulated intracellular cAMPby the CRF antagonist a-helical (9-41) CRF. Each determination is takenfrom a representative experiment performed in triplicate, repeated atleast twice. Cells were pretreated with IBMX. Rat/Human (r/h) CRF wasadded with (solid bars) or without (hollow bars) 2 mM a-helical (9-41).

FIGS. 3A and 3B illustrate a sequence comparison of mouse CRF-RB (SEQ IDNO:10) with mouse CRF-RA (SEQ ID NO:13). The alignment was made usingthe Jotun-Hein method with PAM250 residue weight table. Putativetransmembrane domains are indicated with a solid bar above the sequence.Potential glycosylation sites are indicated by an (*).

FIG. 4 illustrates results from a competitive displacement of¹²⁵I-(Nle21, Tyr32)-ovine CRF bound to membranes from COSM6 cellstransfected with CRF-RB₁. “T” indicates “total hormone” and “B”indicates “bound hormone.” The data are pooled from three independentexperiments.

FIG. 5 illustrates the accumulation of intracellular cAMP in COSM6 cellstransfected with pCRF-RB₁ stimulated by r/hCRF (n), sauvagine (O),suckerfish urotensin (_), hGRF(1-40) OH (o), and VIP (o). Data are alsoshown for the inhibition of stimulation when the cells are exposed to 1mM antagonist (DPhe¹²,Nle^(21,38))hCRF(12-41) (t). The data are from onerepresentative experiment described in Example 9, repeated at leasttwice. The error bars represent the SEM and are smaller than the symbolsif not visible.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a family ofisolated mammalian G-protein-coupled CRF-R proteins characterized ashaving sufficient binding affinity for CRF and CRF-like ligands suchthat concentrations of £ 10 nM of CRF or CRF-like ligands occupy ³50% ofthe binding sites of approximately 0.8 nM of said receptor protein (orapproximately 10-20 pmol receptor/mg membrane protein).

Use of the phrase “isolated” in the present specification and claims asa modifier of DNA, RNA, polypeptides or proteins means that the DNA,RNA, polypeptides or proteins so designated have been produced in suchform by the hand of man, and thus are separated from their native invivo cellular environment. As a result of this human intervention, therecombinant, isolated and/or substantially pure DNAs, RNAs, polypeptidesand proteins of the invention can be produced in large quantities andare useful in ways that the DNAs, RNAs, polypeptides or proteins as theynaturally occur are not, such as identification of selective drugs orcompounds.

As used herein, “mammalian” refers to the variety of species from whichthe invention CRF-R protein is derived, e.g., human, rat, mouse, rabbit,monkey, baboon, bovine, porcine, ovine, canine, feline, and the like.Invention receptors can be derived from a variety of tissue sources,such as, for example, pituitary cells, placental cells, spleen cells,adrenal cells, hematopoietic cells, brain cells, gonadal cells,mesenchymal cells, kidney cells, and the like.

As employed herein, the term “CRF-R” refers to a family of isolatedand/or substantially pure receptor protein subtypes which participate inthe G-protein-coupled response of cells to CRF and CRF-like ligands.Exemplary CRF peptides include r/h CRF and ovine CRF (see U.S. Pat. No.4,415,558), and the like. As employed herein, the phrase “CRF-likeligands” includes substances which have a substantial degree of homology(at least 20% homology) with the amino acid sequence of naturallyoccurring mammalian CRF, as well as alleles, fragments, homologs orderivatives thereof which have substantially the same biologicalactivity as mammalian CRF. Suitable CRF-like ligands can be obtainedfrom a variety of vertebrate species and include such compounds assauvagine (see, e.g., U.S. Pat. No. 4,605,642), urotensin (see, e.g.,U.S. Pat. Nos. 4,908,352; 4,533,654; and 4,525,189) the CRF analogsdescribed in U.S. Pat. Nos.: 4,415,558; 4,489,163; 4,594,329; 4,605,642;5,109,111, each of which are incorporated herein by reference, and thelike.

Such receptor subtypes are typically characterized by having sevenputative transmembrane domains, preceded by a large extracellularamino-terminal domain and followed by a large intracellularcarboxy-terminal domain. Hydropathy analysis of exemplary inventionCRF-Rs (described in SEQ ID NOs: 2, 4, 6 and 10) indicates eighthydrophobic regions of approximately 20 amino acids, corresponding to apossible signal peptide at the N-terminus, plus seven putativetransmembrane domains. After removal of the signal peptide, an exemplaryinvention receptor (as described, for example, in SEQ ID NO:2) has amolecular weight of approximately 40-45 kilodaltons.

Exemplary CRF-R amino acid structures are set forth in SEQ ID NOs 2, 4,6, 8, 10 and 15 of the Sequence Listing provided hereinafter. The CRF-Rdescribed in SEQ ID NO:2 contains five potential glycosylation sites atamino acid positions 38, 45, 78, 90 and 98 (and is referred to herein asCRF-RA₁). Potential protein kinase C phosphorylation sites are locatedin the first and second intracellular loops and in the C-terminal tailat positions 146, 222, 386, and 408. Potential casein kinase II andprotein kinase A phosphorylation sites are located at positions 301 and302, respectively. The third intracellular loop of the invention CRF-Rset forth in SEQ ID NO:2 contains an amino acid sequence similar to theG_(s) activating region found in the third intracellular loop of theb₂-adrenergic receptor.

The invention receptor described in SEQ ID NO:2 exhibits appropriatepharmacologic specificity, i.e., having high affinity for human/rat CRF,ovine CRF, the CRF antagonist a helical (9-41) CRF,(DPhe¹²,Nle^(21,38))hCRF(12-41), urotensins, sauvagine, and very lowaffinity for the biologically impotent analog, [Ala¹⁴]-oCRF. A series ofnon-related peptides are inactive, including such compounds as growthhormone releasing factor, salmon calcitonin, vasoactive intestinalpolypeptide, and gonadotropin releasing hormone, as shown in FIG. 2C.

Binding affinity (which can be expressed in terms of associationconstants, Ka, or dissociation constants, K_(d)) refers to the strengthof interaction between ligand and receptor, and can be expressed interms of the concentration of ligand necessary to occupy one-half (50%)of the binding sites of the receptor. A receptor having a high bindingaffinity for a given ligand will require the presence of very littleligand to become at least 50% bound (hence the K_(d) value will be asmall number); conversely, receptor having a low binding affinity for agiven ligand will require the presence of high levels of ligand tobecome 50% bound (hence the K_(d) value will be a large number).

Reference to receptor protein “having sufficient binding affinity suchthat concentrations of CRF less or CRF-like peptides than or equal to 10nM (i.e., £ 10 nM) occupy ³50% (i.e., greater than or equal to one-half)of the binding sites of said receptor protein” means that ligand (i.e.,CRF) concentration(s) of no greater than about 10 nM are required inorder for the ligand to occupy at least 50% of the active sites ofapproximately 0.8 nM of said receptor (or approximately 10-20 pmolreceptor/mg membrane protein), with much lower ligand concentrationstypically being required. Presently preferred receptors are those whichhave a binding affinity such that ligand concentration(s) in the rangeof only about 1-10 nM are required in order to occupy (or bind to) atleast 50% of the receptor binding sites.

Members of the invention family of receptors can be divided into varioussubclasses, based on the degree of similarity between specific members.For example, genomic sequences encoding CRF receptors of the samesubclass typically have substantially similar restriction maps, whilegenomic sequences encoding CRF receptors of different subclassestypically have substantially different restriction maps. In addition,sequences encoding members of the same subclass of receptors willhybridize under high stringency conditions, whereas sequences encodingmembers of different subclasses will hybridize under low stringencyhybridization conditions, but not under high stringency hybridizationconditions.

Thus, each member of a given subclass is related to other members of thesame subclass by having a high degree of homology (e.g., >80% overallamino acid homology) between specific members; whereas members of agiven subclass differ from members of a different subclass by having alower degree of homology (e.g., about 30% up to 80% overall amino acidhomology) between specific members of different subclasses.

Based on the above criteria, the receptor species described herein canbe designated as CRF-RA or CRF-RB subtypes. Thus, the receptor describedin SEQ ID NO:2 is a CRF-RA subtype, and is referred to herein ashCRF-RA₁ (for human CRF-R, subtype A, variant 1). The modified form ofhCRF-RA₁ which contains the insert sequence set forth in SEQ ID NO:4 isreferred to herein as hCRF-RA₂. Similarly, the receptor described in SEQID NO:6 is referred to herein as rCRF-RA (for rat CRF-R, subtype A), andthe receptor described in SEQ ID NOs:8 and 10 are referred to herein asmCRF-RB₁ (for mouse CRF-R, subtype B, variant 1).

The mouse CRF-RA₁ and CRF-RB₁ receptors have been compared and are seento be about 70% homologous at the nucleotide level and 68% homologous atthe amino acid level (see, e.g., FIGS. 3A and 3B). In addition, thereare a number of fundamental structural characteristics that areconserved between the two receptors. The number and location ofpotential N-glycosylation sites are the same. Six cysteines are presentin the N-terminal domain, characteristic of the receptor family. In theCRF-RB₁ receptor, however, there are two additional cysteines, one inthe N-terminus and the other at the junction of the first extracellularloop (i.e., ECL-1) and the third transmembrane domain (i.e., TMD-3). Ifone assumes that the N-terminal cysteine is removed with the signalpeptide and that the latter cysteine is within the transmembrane domain,CRF-RB₁ is seen to have six cysteines in the extracellular region, as isthe case for other members of the receptor family.

The first intracellular loop is practically the same between CRF-RA₁ andCRF-RB₁, except for the substitution of a valine for the arginine foundin CRF-RA₁. The second intracellular loop differs in three amino acids,but the changes are conservative. Thus, a methionine, a glutamic acidand a histidine are present in CRF-RB₁, instead of a leucine, anaspartic acid and an arginine, respectively, in CRF-RA₁. The thirdintracellular loop is 100% identical between the two receptors. TheC-terminal domain is also highly conserved between the two receptors.The putative phosphorylation sites in the intracellular loops are alsonearly identical between CRF-RA₁ and CRF-RB₁, with one exceptionoccurring in the C-terminus, in which SER386 in CRF-RA₁ is present as analanine in CRF-RB₁.

A major determinant of the coupling invention CRF-receptors toGTP-binding proteins and subsequently to adenylate cyclase is thought toreside in the third intracellular loop and the C-terminus. Because ofthe high similarity of the third intracellular loop and the C-termini ofCRF-RA₁ and CRF-RB₁, the coupling and signal transduction properties ofthe two receptors are expected to be very similar. Indeed, the data(FIG. 5) demonstrate that the signal transduction characteristics ofCRF-RA₁ and CRF-RB₁ are nearly identical. It is expected that a moredetailed analysis of the desensitization characteristics of the tworeceptors will reveal subtle differences as a consequence of the changesin the C-terminus and the other intracellular loops.

The main differences between the two receptors are found in theN-terminal domain, in which sixteen extra amino acids are found inCRF-RB₁, and in which there are significant, non-conservative amino acidchanges in the remaining portion of the N-terminus. It is interesting tonote that, based on the genomic sequence of the mouse CRF-RA₁, the aminoacid sequences of the two receptors start to diverge very close to thesecond intron/exon junction in the N-terminus, raising the possibilitythat some of the divergence between the two receptors could result fromalternative exon utilization (i.e., splice variants). Indeed, thepresence of multiple protected RNA species when using N-terminal probesis consistent with the existence of splice variants of this receptor.

In addition to the N-terminal domain, the first, second, and thirdextracellular loops (ECLs-1, -2, and -3) also contain significantdifferences. For example, extracellular loop-1 in CRF-RB₁ contains morecharged residues than does the corresponding loop in CRF-RA₁.Extracellular loop-2 in CRF-RA₁ contains an arginine, instead of aglutamic acid in CRF-RB₁. Extracellular loop-3 is the most similarbetween the two receptors. It is presently believed that a major bindingdeterminant in this family of receptors is the N-terminal region and theextracellular loops. Therefore, the existence of differences in theextracellular domains suggests that the binding specificities betweenthe two receptors should differ. Urotensin (K_(i)=0.7±0.3, n=3) andsauvagine (K_(i)=0.6±0.1, n=3) show a trend to be more potent thanr/hCRF (K_(i)=1.3±0.2, n=6) on CRF-RB.

In situ hybridization studies indicate that mRNAs related to CRF-RB havea restricted distribution in the central nervous system that differsconsiderably from that of CRF-RA. Thus, the receptors derived from thetwo genes, CRF-RA and CRF-RB, with their distinct tissue distributionsand structural diversity especially in the extracellular domains arelikely to subserve disparate biological roles.

It is expected that a more detailed pharmacological comparison of thetwo receptors is likely to reveal significant differences in the bindingcharacteristics of the two receptors. It is also expected that differentCRF-R subtypes will mediate different actions of CRF. Thus, by havingavailable nucleic acid encoding various CRF-R subtypes, those of skillin the art have been enabled to screen for and develop selective analogsspecific for each CRF-R subtype. The analogs so obtained will be morespecific, potent, and effective at binding and modulating the activityof the respective CRF-R subtype.

In one embodiment of the present invention, the CRF-RA₁ encoded by theclone referred to herein as “hctCRFR” (described hereinafter) has a highbinding affinity for r/h CRF [K_(d)=3.3±0.45 nM (n=4)]; ovine CRF[K_(d)=2.3±0.66 nM (n=3)]; and for the antagonist a helCRF(9-41)[K_(d)=13.0±5.2 nM (n=3)]. This receptor has a low binding affinity forthe biologically impotent analog, [Ala¹⁴]-ovine CRF [K_(d)>300 nM (n=2)]. In another embodiment of the present invention, the CRF-R describedin SEQ ID NO:2 has a binding affinity for r/h CRF of K_(d)=3.8±0.20 nM,(n=1).

Presently preferred receptor proteins of the invention have amino acidsequences that are substantially the same as the sequences set forth inSequence ID Nos. 2, 4, 6, 8, and 10 and amino acid sequences which aresubstantially the same as the amino acid sequences encoded by theCRF-RA₁-encoding portion of clone hctCRFR, deposited with the ATCC underaccession number 75474, as well as functional, modified forms thereof.Those of skill in the art recognize that numerous residues of theabove-described sequences can be substituted with other, chemically,sterically and/or electronically similar residues without substantiallyaltering the biological activity of the resulting receptor species.

The htcCRFR clone was deposited Jun. 2, 1993, at the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., U.S.A.20852, under the terms of the Budapest Treaty on the InternationalRecognition of Deposits of Microorganisms for Purposes of PatentProcedure and the Regulations promulgated under this Treaty. Samples ofthe deposited material are and will be available to industrial propertyoffices and other persons legally entitled to receive them under theterms of the Treaty and Regulations and otherwise in compliance with thepatent laws and regulations of the United States of America and allother nations or international organizations in which this application,or an application claiming priority of this application, is filed or inwhich any patent granted on any such application is granted. Inparticular, upon issuance of a U.S. patent based on this or anyapplication claiming priority to or incorporating this application byreference thereto, all restriction upon availability of the depositedmaterial will be irrevocably removed.

As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological properties characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95% aminoacid sequence identity being especially preferred.

Recombinant CRF-R protein can be routinely obtained, employing theinvention nucleic acids described hereinafter, having significantlyhigher purity than naturally occurring CRF-R (e.g., substantially freeof other proteins present in crude extracts from mammalian cells).Recombinant DNA techniques well-known in the art, for example, can beused to generate organisms or cell lines that produce heterologous CRF-Rprotein in significantly higher purities, relative to naturallyoccurring membrane protein. Subsequently, using appropriate isolationtechniques, it is possible to routinely obtain CRF-R proteins which areat least about 70%, preferably 80%, more preferably 90%, and mostpreferred 98% pure (by weight of total proteins), and which is hereinreferred to as substantially pure.

In accordance with a further embodiment of the present invention, thereis provided a binding assay employing receptors of the invention,whereby a large number of compounds can be rapidly screened to determinewhich compounds, if any, are capable of binding to the receptors of theinvention. Subsequently, more detailed assays can be carried out withinitially identified compounds, to further determine whether suchcompounds act as agonists or antagonists of invention receptors.

Another application of the binding assay of the invention is the assayof test samples (e.g., biological fluids) for the presence or absence ofCRF. Thus, for example, serum from a patient displaying symptoms thoughtto be related to over- or under-production of CRF can be assayed todetermine if the observed symptoms are indeed caused by over- orunder-production of CRF (or CRF receptor).

The binding assays contemplated by the present invention can be carriedout in a variety of ways, as can readily be identified by one of skillin the art. For example, competitive binding assays can be employed, aswell as radioimmunoassays, ELISA, ERMA, and the like.

In accordance with a still further embodiment of the present invention,there are provided bioassays for evaluating whether test compounds arecapable of acting as agonists or antagonists of receptor(s) of thepresent invention (or functional modified forms thereof).

Invention CRF-Rs are coupled by heterotrimeric G-proteins to variousintracellular enzymes, ion channels, and transporters. The G-proteinsassociate with invention CRF-R proteins at the intracellular face of theplasma membrane. An agonist binding to CRF-R catalyzes the exchanges ofGTP for GDP on the a-subunit (G-protein “activation”), resulting in itsdissociation and stimulation of one (or more) of the varioussignal-transducing enzymes and channels. The different G-proteina-subunits preferentially stimulate particular effectors. Thespecificity of signal transduction may be determined, therefore, by thespecificity of G-protein coupling.

It has been found that invention CRF-R proteins mediate signaltransduction through the modulation of adenylate cyclase. For example,when CRF binds to CRF-R, adenylate cyclase causes an elevation in thelevel of intracellular cAMP. Accordingly, in one embodiment of thepresent invention, the bioassay for evaluating whether test compoundsare capable of acting as agonists or antagonists comprises:

-   -   (a) culturing cells containing:        -   DNA which expresses CRF receptor protein(s) or functional            modified forms thereof,        -   wherein said culturing is carried out in the presence of at            least one compound whose ability to modulate signal            transduction activity of CRF receptor protein is sought to            be determined, and thereafter    -   (b) monitoring said cells for either an increase or decrease in        the level of intracellular cAMP.

Methods well-known in the art that measure intracellular levels of cAMP,or measure cyclase activity, can be employed in binding assays describedherein to identify agonists and antagonists of the CRF-R. For example,because activation of some G-protein-coupled receptors results indecreases or increases in cAMP, assays that measure intracellular cAMPlevels (see, e.g., Example 4) can be used to evaluate recombinant CRF-Rsexpressed in mammalian host cells.

As used herein, “ability to modulate signal transduction activity of CRFreceptor protein” refers to a compound that has the ability to eitherinduce or inhibit signal transduction activity of the CRF receptorprotein.

In another embodiment of the present invention, the bioassay forevaluating whether test compounds are capable of acting as agonistscomprises:

-   -   (a) culturing cells containing:        -   DNA which expresses CRF receptor protein(s) or functional            modified forms thereof, and        -   DNA encoding a reporter protein, wherein said DNA is            operatively linked to a CRF-R responsive transcription            element;        -   wherein said culturing is carried out in the presence of at            least one compound whose ability to induce signal            transduction activity of CRF receptor protein is sought to            be determined, and thereafter    -   (b) monitoring said cells for expression of said reporter        protein.

In another embodiment of the present invention, the bioassay forevaluating whether test compounds are capable of acting as antagonistsfor receptor(s) of the invention, or functional modified forms of saidreceptor(s), comprises:

-   -   (a) culturing cells containing:        -   DNA which expresses CRF receptor protein(s), or functional            modified forms thereof, and        -   DNA encoding a reporter protein, wherein said DNA is            operatively linked to a CRF-R responsive transcription            element        -   wherein said culturing is carried out in the presence of:            -   increasing concentrations of at least one compound whose                ability to inhibit signal transduction activity of CRF                receptor protein(s) is sought to be determined, and            -   a fixed concentration of at least one agonist for CRF                receptor protein(s), or functional modified forms                thereof; and thereafter    -   (b) monitoring in said cells the level of expression of said        reporter protein as a function of the concentration of said        compound, thereby indicating the ability of said compound to        inhibit signal transduction activity.

In step (a) of the above-described antagonist bioassay, culturing mayalso be carried out in the presence of:

-   -   fixed concentrations of at least one compound whose ability to        inhibit signal transduction activity of CRF receptor protein(s)        is sought to be determined, and    -   an increasing concentration of at least one agonist for CRF        receptor protein(s), or functional modified forms thereof.

Host cells for functional recombinant expression of CRF-Rs preferablyexpress endogenous or recombinant guanine nucleotide-binding proteins(i.e., G-proteins). G-proteins are a highly conserved family ofmembrane-associated proteins composed of a, b and g subunits. The asubunit, which binds GDP and GTP, differs in different G-proteins. Theattached pair of b and g subunits may or may not be unique; different achains may be linked to an identical bg pair or to different pairs[Linder and Gilman, Sci. Am. 267:56-65 (1992)]. More than 30 differentcDNAs encoding G protein a subunits have been cloned [Simon et al.,Science 252:802 (1991)]. At least four different b polypeptide sequencesare known [Simon et al., Science 252:802 (1991)]. G-proteins switchbetween active and inactive states by guanine nucleotide exchange andGTP hydrolysis. Inactive G protein is stimulated by a ligand-activatedreceptor to exchange GDP for GTP. In the active form, the a subunit,bound to GTP, dissociates from the bg complex, and the subunits theninteract specifically with cellular effector molecules to evoke acellular response. Because different G-proteins can interact withdifferent effector systems (e.g., phospholipase C, adenyl cyclasesystems) and different receptors, it is useful to investigate differenthost cells for expression of different recombinant CRF-R receptorsubtypes. Alternatively, host cells can be transfected with G-proteinsubunit-encoding DNAs for heterologous expression of differing Gproteins.

Host cells contemplated for use in the bioassay(s) of the presentinvention include CV-1 cells, COS cells, and the like; reporter andexpression plasmids employed typically also contain the origin ofreplication of SV-40; and the reporter and expression plasmids employedalso typically contain a selectable marker.

As used herein, a “CRF-R responsive transcription element” is anypromoter region that is induced, e.g., by the well-known G-proteinmediated signal transduction mechanism, to initiate transcription uponthe binding of a CRF-R agonist, such as CRF. A preferred CRF-Rresponsive transcription element is a cAMP responsive transcriptionelement. Cyclic AMP (cAMP) responsive transcription elements employed inthe bioassay(s) of the present invention are well-known to those ofskill in the art. The cAMP response elements respond to increases inintracellular cAMP by initiating trascription of the DNA molecule (i.e.,a reporter gene) operatively linked thereto. An exemplary cAMP responseelement suitable for use herein is the human DNA b-Polymerase genepromoter (see Mamula et al., DNA and Cell Bio., 11:61-70, 1992).

Reporter proteins suitable for use herein are well known in the art.Host cells can be monitored for the level of expression of a reportergene encoding a reporter protein in a variety of ways, such as, forexample, by photometric means, e.g., by colorimetry (with a coloredreporter product such as β-galactosidase), by fluorescence (with areporter product such as luciferase), by enzyme activity, and the like.

Compounds contemplated for screening in accordance with the inventionbioassays include CRF or CRF-like ligands, as well as compounds whichbear no particular structural or biological relatedness to CRF. Suitablecompounds may be obtained from well-known sources, e.g., from peptidelibraries, chemical libraries, bacterial and yeast broths, plants, andthe like.

Examples of compounds which bear no particular structural or biologicalrelatedness to CRF, but which are contemplated for screening inaccordance with the bioassays of the present invention, include anycompound that is an antagonist (i.e., is capable of blocking the actionof the invention receptor peptides), or an agonist (i.e., is capable ofpromoting the action of the invention receptor peptides), such as, forexample, alkaloids and other heterocyclic organic compounds, and thelike.

As employed herein, the term “non-CRF-like” proteins refers to anyorganic molecule having essentially no structural similarity with CRF(as defined broadly herein).

Also encompassed by the term CRF-R are the various subtypes thereof(e.g., CRF-RA (such as hCRF-RA₁, and hCRF-RA₂), CRF-RB₁, and the like),as well as polypeptide fragments or analogs thereof. Therefore, a CRF-Rcontemplated by the present invention can be subject to various changes,substitutions, insertions, and deletions, where such changes provide forcertain advantages in its use. For example, a peptide fragment iscapable of immunologically mimicking a CRF-R native antigenic epitope oris capable of exhibiting another biological property characteristic ofCRF-R, such as, for example, binding to CRF or binding to G-protein(s).

Specific CRF-R residues or regions which are necessary for efficientsignal transduction may interact with conserved G-protein motifs. Inaddition, certain short amino acid stretches of the CRF-R, which arenecessary for G-protein coupling, also determine the specificity of theG-protein interactions. Thus, polypeptide fragments of the inventionCRF-R are useful in assays or therapeutic methods in which controlledbinding to various G-proteins is desired.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the ability to mimicCRF-R as described herein. Examples of conservative substitutionsinclude the substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue,provided that such polypeptide displays the requisite binding activity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include, for example, those molecules inwhich free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For examples: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions of residues, relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

When additional residues have been added at either terminus for thepurpose of providing a “linker” by which the polypeptides of theinvention can be conveniently affixed to a label or solid matrix, orcarrier, the linker residues do not form CRF-R epitopes, i.e., are notsimilar in structure to CRF-R. Labels, solid matrices, and carriers thatcan be used with the polypeptides of this invention are describedhereinbelow.

Amino acid residue linkers include at least one residue up to 40 or moreresidues (more often they comprise 1 to 10 residues), but do not formCRF-R epitopes. Typical amino acid residues used for linking aretyrosine, cysteine, lysine, glutamic acid and aspartic acid. Inaddition, a subject polypeptide can differ in sequence, unless otherwisespecified, from the natural sequence of CRF-R by modification of thesequence by N-terminal acylation e.g., acetylation or thioglycolic acidamidation, and by C-terminal amidation, e.g., with ammonia, methylamine,and the like.

CRF-R polypeptides of the present invention are capable of inducingantibodies that immunoreact with CRF-R. In view of the well establishedprinciple of immunologic cross-reactivity, the present inventiontherefore contemplates antigenically related variants of thepolypeptides. An “antigenically related variant” is a subjectpolypeptide that is capable of inducing antibody molecules thatimmunoreact with the CRF-R polypeptides described herein.

CRF-R polypeptides of the present invention can be synthesized by any ofthe techniques that are known to those skilled in the polypeptide art,including recombinant DNA techniques. Synthetic chemistry techniques,such as solid-phase Merrifield-type synthesis, are preferred forproducing polypeptide fragments for reasons of purity, antigenicspecificity, freedom from undesired side products, ease of production,and the like. An excellent summary of the many techniques available canbe found in J. M. Steward and J. D. Young, “Solid Phase PeptideSynthesis”, W. H. Freeman Co., San Francisco, 1969; M. Bodansky, et al.,“Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976 and J.Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (New York), 1983 for solid phase peptide synthesis, and E.Schroder and K. Kubke, “The Peptides”, Vol. 1, Academic Press (NewYork), 1965 for classical solution synthesis, each of which isincorporated herein by reference. Appropriate protective groups usablein such synthesis are described in the above texts and in J. F. W.McOmie, “Protective Groups in Organic Chemistry”, Plenum Press, NewYork, 1973, which is incorporated herein by reference. See also U.S.Pat. No. 5,055,396, incorporated herein by reference.

CRF-R polypeptides can be used, inter alia, in diagnostic methods andsystems according to the present invention to detect the level of CRF-R(or fragments thereof) present in a body sample, to detect the level ofCRF in a body sample, or to prepare an inoculum as described herein forthe preparation of antibodies that immunoreact with epitopes on CRF-R.CRF-R polypeptides can also be used to bind, detect and purify variousintracellular G-proteins and CRF-like receptor agonist/antagonists, suchas heterocyclic compounds, and the like. In addition, CRF-R polypeptidescan be used in therapeutic methods described herein, e.g., to inhibitthe CRF-induced ACTH release and decrease the level of ACTH in apatient.

In accordance with yet another embodiment of the present invention,there are provided antibodies generated against the above-describedreceptor proteins. Such antibodies can be employed for diagnosticapplications, therapeutic applications, and the like. Preferably, fortherapeutic applications, the antibodies employed will be monoclonalantibodies.

The above-described antibodies can be prepared employing standardtechniques, as are well known to those of skill in the art, usinginvention receptor proteins, or fragments thereof, as antigens forantibody production. Antibodies of the present invention are typicallyproduced by immunizing a mammal with an inoculum containing a CRF-Rprotein or fragment thereof thereby inducing the production of antibodymolecules having immunospecificity for the immunizing agent.

For example, antibodies raised in rabbits against a synthetic peptidefragment of the invention protein recognize the synthetic peptide andthe corresponding invention CRF-R on an equimolar basis, and preferably,are capable of inhibiting the activity of the native protein. Antibodiesto CRF-R may be obtained, for example, by immunizing three month oldmale and female white New Zealand rabbits with a suitable syntheticpeptide fragment to which Tyr has been added at the C-terminus in orderto couple it, as an antigen, to BSA by a bisdiazotized benzidine (BDB)linkage by reaction for 2 hours at 4° C. The reaction mixture isdialyzed to remove low molecular weight material, and the retentate isfrozen in liquid nitrogen and stored at −20° C. Animals are immunizedwith the equivalent of 1 mg of the peptide antigen according to theprocedure of Vaughan et al., Meth. in Enzymology, 168:588-617 (1989). Atfour week intervals, the animals are boosted by injections of 200 mg ofthe antigen and bled ten to fourteen days later. After the third boost,antiserum is examined for its capacity to bind radioiodinated antigenpeptide prepared by the chloramine-T method and then purified by CMC-ionexchange column chromatography or HPLC. The antibody molecules are thencollected from the mammal and isolated to the extent desired by wellknown techniques such as, for example, by using DEAE Sephadex to obtainthe IgG fraction.

To enhance the specificity of the antibody, the antibodies may bepurified by immunoaffinity chromatography using solid-phase immunizingpolypeptide. The antibody is contacted with the solid-phase immunizingpolypeptide for a period of time sufficient for the polypeptide toimmunoreact with the antibody molecules to form a solid-phaseimmunocomplex. The bound antibodies are separated from the complex bystandard techniques.

Antibody so produced can be used, inter alia, in diagnostic methods andsystems to detect the level of CRF-R present in a mammalian, preferablyhuman, body sample, such as tissue or vascular fluid. The anti-CRF-Rantibodies can also be used for immunoaffinity or affinitychromatography purification of CRF-R biological materials. In addition,an anti-CRF-R antibody according to the present invention can be used inmammalian therapeutic methods, preferably human, as a CRF-R agonist orantagonist, to neutralize or modulate the effect of CRF-R, increase thelevel of free CRF (e.g., CRF not bound by CRF-R), increase CRF-inducedACTH release, increase the level of ACTH-induced glucocorticoids in apatient, and the like.

The proteins of the invention, and the antibodies of the invention, canbe administered to a subject employing standard methods, such as, forexample, by intraperitoneal, intramuscular, intravenous, or subcutaneousinjection, and the like. Implant and transdermal modes of administrationare also appropriate. In addition, proteins of the invention can bedelivered by transfection with viral or retroviral vectors that encodeinvention protein. One of skill in the art can readily determine doseforms, treatment regiments, etc, depending on the mode of administrationemployed.

In accordance with a further embodiment of the present invention, thereare provided isolated and purified nucleic acid molecules (e.g., DNA orRNA) which encode the above-described receptor proteins. The nucleicacid molecules described herein are useful for producing invention CRF-Rproteins, when such nucleic acids are incorporated into a variety ofprotein expression systems known to those of skill in the art. Inaddition, such nucleic acid molecules (or fragments thereof) can belabeled with a readily detectable substituent and used as hybridizationprobes for assaying for the presence and/or amount of a CRF-R gene ormRNA transcript in a given sample. Such nucleic acid molecules (orfragments thereof), when labeled with a readily detectable substituent,can also be used as hybridization probes for identifying additionalCRF-R genes. The nucleic acid molecules described herein, and fragmentsthereof, are also useful as primers and/or templates in a PCR reactionfor amplifying genes encoding the CRF-R protein described herein. Inaddition, the nucleic acid molecules described herein, and fragmentsthereof, are also useful as primers and/or templates in a PCR reactionfor identifying genes encoding additional CRF-R proteins which are partof the family of receptor proteins described herein.

The above-described receptor(s) can be encoded by numerous nucleic acidmolecules, e.g., a nucleic acid molecule having a contiguous nucleotidesequence substantially the same as:

-   -   nucleotides 82-1329 of Sequence ID No. 1,    -   nucleotides 82-1329 of Sequence ID No. 1, further containing        nucleotides 1-87 of SEQ ID No. 3 inserted between nucleotides        516-517 of SEQ ID No. 1,    -   nucleotides 81-1324 of Sequence ID No. 5,    -   substantially all nucleotides of Sequence ID No. 7,    -   nucleotides 79-1371 of Sequence ID No. 9,    -   the CRF-RA₁-encoding portion of clone hctCRFR, deposited with        the ATCC under accession number 75474,    -   or variations thereof which encode the same amino acid        sequences, but employ different codons for some of the amino        acids,    -   or splice variant cDNA sequences thereof.

As employed herein, the phrase “nucleic acid” refers to ribonucleic acid(RNA) or deoxyribonucleic acid (DNA). DNA can be either complementaryDNA (cDNA) or genomic DNA, e.g. a gene encoding a CRF-R.

As employed herein, the phrases “contiguous nucleotide sequencesubstantially the same as” or “substantially the same nucleotidesequence” refers to DNA having sufficient homology to the referencepolynucleotide, such that it will hybridize to the reference nucleotideunder typical moderate stringency conditions. In one embodiment, nucleicacid molecules having substantially the same nucleotide sequence as thereference nucleotide sequence encodes substantially the same amino acidsequence as that of any one of SEQ ID NOs:2, 4, 6, 8, or 10. In anotherembodiment, DNA having “substantially the same nucleotide sequence” asthe reference nucleotide sequence has at least 60% homology with respectto the reference nucleotide sequence. DNA having at least 70%, morepreferably 80%, yet more preferably 90%, homology to the referencenucleotide sequence is preferred.

Yet other DNAs which encode the above-described receptor are thosehaving a contiguous nucleotide sequence substantially the same as setforth in Sequence ID Nos. 1, 3, 5, 7, or 9 or the CRF-RA₁-encodingportion of clone hctCRFR, deposited with the ATCC under accession number75474.

“Gene(s)” (i.e., genomic DNA) encoding invention CRF-Rs typicallycontain at least two introns. Thus, alternatively spliced variant cDNAsequences encoding invention CRF-Rs are contemplated herein (e.g.,CRF-RA₂). For example, SEQ ID NO:3 sets forth an 87 bp cDNA splicevariant insert sequence that is inserted between nucleotide positions516-517 of the CRF-RA₁, encoding cDNA set forth in SEQ ID NO:1 (therebyproducing CRF-RA₂).

As used herein, the phrases “splice variant” or “alternatively spliced”,when used to describe a particular nucleotide sequence encoding aninvention receptor, refers to a cDNA sequence that results from the wellknown eukaryotic RNA splicing process. The RNA splicing process involvesthe removal of introns and the joining of exons from eukaryotic primaryRNA transcripts to create mature RNA molecules of the cytoplasm.

Methods of isolating splice variant nucleotide sequences are well knownin the art. For example, one of skill in the art can employ nucleotideprobes derived from the CRF-R encoding cDNA of SEQ ID NOs 1, 3, 5, 7, or9 to screen the Cushing's tumor cDNA library described in the Examplesor other cDNA libraries derived from cells believed to express CRF-Rs,e.g., brain, heart, pituitary, immune, gonadal, adrenal, placental,gastrointestinal, pulmonary, corticotropic, and the like.

In a preferred embodiment, cDNA encoding CRF-Rs as disclosed herein havesubstantially the same nucleotide sequence as nucleotides 82-1329 of SEQID NO:1, as nucleotides 82-1329 of SEQ ID NO:1 further containingnucleotides 1-87 of SEQ ID NO:3 inserted between nucleotides 516-517 ofSEQ ID NO:1, as SEQ ID NO:5, as nucleotides 81-1324 of SEQ ID NO:7, oras nucleotides 79-1371 of SEQ ID NO:9. The presently most preferred cDNAmolecules encoding the CRF-Rs have the same nucleotide sequence asnucleotides 82-1329 of SEQ ID NO:1, as nucleotides 82-1329 of SEQ IDNO:1 further containing nucleotides 1-87 of SEQ ID NO:3 inserted betweennucleotides 516-517 of SEQ ID NO:1, as SEQ ID NO:5, as nucleotides81-1324 of SEQ ID NO:7, or as nucleotides 79-1371 of SEQ ID NO:9.

In accordance with another embodiment of the present invention, isolatedand purified nucleic acid encoding a CRF-R may be selected from:

-   -   (a) DNA encoding the amino acid sequence set forth in SEQ ID        NO:2, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10; or DNA encoding        the amino acid sequence set forth in SEQ ID NO:2 further        comprising the amino acid sequence set forth in SEQ ID NO:4        inserted between amino acids 145-146 of SEQ ID NO:2, or    -   (b) DNA that hybridizes to the DNA of (a) under moderately        stringent conditions, wherein said DNA encodes biologically        active CRF-R, or    -   (c) DNA degenerate with respect to either (a) or (b) above,        wherein said DNA encodes biologically active CRF-R.

Hybridization refers to the binding of complementary strands of nucleicacid (i.e., sense:antisense strands or probe:target-DNA) to each otherthrough hydrogen bonds, similar to the bonds that naturally occur inchromosal DNA. Stringency levels used to hybridize a given probe withtarget-DNA can be readily varied by those of skill in the art.

As used herein, the phrase “moderately stringent” hybridization refersto conditions that permit target-DNA to bind a complementary nucleicacid that has about 60%, preferably about 75%, more preferably about85%, homology to the target DNA; with greater than about 90% homology totarget-DNA being especially preferred. Preferably, moderately stringentconditions are conditions equivalent to hybridization in 50% formamide,5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in0.2×SSPE, 0.2% SDS, at 65° C. Denhart's solution and SSPE (see, e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1989) are well known to those of skill in theart as are other suitable hybridization buffers.

The term “functional” or “biologically active”, when used herein as amodifier of receptor protein(s) of the present invention, refers to apolypeptide that is able to produce one of the functionalcharacteristics, e.g., antigenicity, exhibited by any of the CRF-Rsdescribed herein. In another embodiment, biologically active means thatbinding of CRF-like ligands (such as CRF analogs, urotensin, sauvagine,and the like) to said receptor protein(s) modifies the receptorinteraction with G-proteins, which in turn affects the levels ofintracellular second messengers, preferably cAMP, leading to a varietyof physiological effects. Stated another way, “functional” means that asignal is transduced as a consequence of agonist activation of receptorprotein(s).

As used herein, the term “degenerate” refers to codons that differ in atleast one nucleotide from a reference nucleic acid, e.g., SEQ ID NO:1,but encode the same amino acids as the reference nucleic acid. Forexample, codons specified by the triplets “UCU”, “UCC”, “UCA”, and “UCG”are degenerate with respect to each other since all four of these codonsencode the amino acid serine.

The invention nucleic acids can be produced by a variety of methodswell-known in the art, e.g., the methods described in Examples 1 and 5,employing PCR amplification using oligonucleotide primers from variousregions of SEQ ID NOs:1, 3, 5, 7, 9 and 14 and the like.

One method employed for isolating and cloning nucleic acids encoding thereceptor(s) of the present invention involves expressing, in mammaliancells, a cDNA library prepared from any cell type thought to respond toCRF (e.g., pituitary cells, placental cells, fibroblast cells, and thelike) in a suitable host cell, such as, for example, COSM6 cells. Theability of the resulting mammalian cells to bind a labeled receptorligand (i.e., a labeled CRF analog) is then determined. Finally, thedesired cDNA insert(s) are recovered, based on the ability of aparticular cDNA, when expressed in mammalian cells, to induce (orenhance) the binding of labeled receptor ligand to said cell.

Alternatively, DNA libraries may be screened employing an immunologicalexpression assay with an antibody raised against the protein ofinterest. Screening of the expression library with antibodies raisedagainst the protein of interest may also be used, alone or inconjunction with hybridization probing, to identify or confirm thepresence of the sought-after DNA sequences in DNA library clones. Suchtechniques are taught, for example, in Maniatis et al., Cold SpringHarbor Laboratory Manual, Cold Spring Harbor, N.Y. (1982), (hereinafterCSH).

In accordance with a further embodiment of the present invention,optionally labeled receptor-encoding cDNAs, or fragments thereof, can beemployed to probe library(ies) (e.g., cDNA, genomic, and the like) foradditional nucleotide sequences encoding novel mammalian members of theCRF receptor family. Such screening is initially carried out underlow-stringency conditions, which comprise a temperature of less thanabout 42.5° C., a formamide concentration of less than about 50%, and amoderate to low salt concentration. Presently preferred screeningconditions comprise a temperature of about 42.5° C., a formamideconcentration of about 20%, and a salt concentration of about 5×standardsaline citrate (SSC; 20×SSC contains 3M sodium chloride, 0.3M sodiumcitrate, pH 7.5). Such conditions will allow the identification ofsequences which have a substantial degree of similarity with the probesequence, without requiring perfect homology for the identification of astable hybrid. The phrase “substantial similarity” refers to sequenceswhich share at least 50% homology. Preferably, hybridization conditionswill be selected which allow the identification of sequences having atleast 70% homology with the probe, while discriminating againstsequences which have a lower degree of homology with the probe.

As used herein, a nucleic acid “probe” is single-stranded DNA or RNA, oranalogs thereof, that has a sequence of nucleotides that includes atleast 14, preferably at least 20, more preferably at least 50,contiguous bases that are the same as (or the complement of) any 14 ormore contiguous bases set forth in any of SEQ ID NOs: 1, 3, 5, 7 or 9 orthe CRF-RA₁-encoding portion of clone hctCRFR. Preferred regions fromwhich to construct probes include 5′ and/or 3′ coding sequences,sequences predicted to encode transmembrane domains, sequences predictedto encode cytoplasmic loops, signal sequences, ligand binding sites, andthe like. The entire cDNA molecule encoding an invention CRF-R may alsobe employed as a probe. Probes may be labeled by methods well-known inthe art, as described hereinafter, and used in various diagnostic kits.

In accordance with yet another embodiment of the present invention,there is provided a method for the recombinant production of inventionreceptor(s) by expressing the above-described nucleic acid sequences insuitable host cells. The above-described nucleotide sequences can beincorporated into vectors for further manipulation. As used herein,vector (or plasmid) refers to discrete elements that are used tointroduce heterologous DNA (e.g., SEQ ID NOs:1, 3, 5, 7 or 9) into cellsfor either expression or replication thereof. Selection and use of suchvehicles are well within the skill of the artisan.

An expression vector includes elements capable of expressing DNAs thatare operatively linked with regulatory sequences (such as promoterregions) that are capable of regulating expression of such DNAfragments. Thus, an expression vector refers to a recombinant DNA or RNAconstruct, such as a plasmid, a phage, recombinant virus or other vectorthat, upon introduction into an appropriate host cell, results inexpression of the cloned DNA. Appropriate expression vectors are wellknown to those of skill in the art and include those that are replicablein eukaryotic cells and/or prokaryotic cells and those that remainepisomal or those which integrate into the host cell genome. Presentlypreferred plasmids for expression of invention CRF-Rs in eukaryotic hostcells, particularly mammalian cells, include cytomegalovirus (CMV)promoter-containing vectors, SV40 promoter-containing vectors, MMTV LTRpromoter-containing vectors, and the like.

As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Thisportion of the promoter region is referred to as the promoter. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

As used herein, the term “operatively linked” refers to the functionalrelationship of DNA with regulatory and effector sequences ofnucleotides, such as promoters, enhancers, transcriptional andtranslational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove, add or alter 5′ untranslated portions of theclones to eliminate extra, potentially inappropriate alternativetranslation initiation (i.e., start) codons or other sequences that mayinterfere with or reduce expression, either at the level oftranscription or translation. Alternatively, consensus ribosome bindingsites (see, for example, Kozak (1991) J. Biol. Chem. 266:19867-19870)can be inserted immediately 5′ of the start codon and may enhanceexpression. The desirability of (or need for) such modification may beempirically determined.

As used herein, expression refers to the process by which polynucleicacids are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleic acid is derived fromgenomic DNA, expression may, if an appropriate eukaryotic host cell ororganism is selected, include splicing of the mRNA.

Prokaryotic transformation vectors are well-known in the art and includepBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla, Calif.),and the like. Other suitable vectors for transformation of E. coli cellsinclude the pET expression vectors (Novagen, see U.S. Pat. No.4,952,496), e.g., pET11a, which contains the T7 promoter, T7 terminator,the inducible E. coli lac operator, and the lac repressor gene; and pET12a-c, which contain the T7 promoter, T7 terminator, and the E. coliompT secretion signal. Another suitable vector is the pIN-IIIompA2 (seeDuffaud et al., Meth. in Enzymology, 153:492-507, 1987), which containsthe 1 pp promoter, the lacUV5 promoter operator, the ompA secretionsignal, and the lac repressor gene.

Particularly preferred base vectors for transfection of mammalian cellsare cytomegalovirus (CMV) promoter-based vectors such as pcDNA1(Invitrogen, San Diego, Calif.), MMTV promoter-based vectors such aspMAMNeo (Clontech, Palo Alto, Calif.) and pMSG (Catalog No. 27-4506-01from Pharmacia, Piscataway, N.J.), and SV40 promoter-based vectors suchas pSVb (Clontech, Palo Alto, Calif.), and the like.

The use of a wide variety of organisms has been described for therecombinant production of proteins or biologically active fragmentsthereof. One of skill in the art can readily determine suitable hosts(and expression conditions) for use in the recombinant production of thepeptides of the present invention. Yeast hosts, bacterial hosts,mammalian hosts, and the like can be employed.

In accordance with another embodiment of the present invention, thereare provided “recombinant cells” containing the nucleic acid molecules(i.e., DNA or mRNA) of the present invention (e.g., SEQ ID NOs:1, 3, 5,7 or 9). Methods of transforming suitable host cells, as well as methodsapplicable for culturing said cells containing a gene encoding aheterologous protein, are generally known in the art. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989).

Exemplary methods of transformation include, e.g., transformationemploying plasmids, viral, or bacterial phage vectors, transfection,electroporation, lipofection, and the like. The heterologous nucleicacid can optionally include sequences which allow for itsextrachromosomal maintenance, or said heterologous nucleic acid can becaused to integrate into the genome of the host (as an alternative meansto ensure stable maintenance in the host).

Host organisms contemplated for use in the practice of the presentinvention include those organisms in which recombinant production ofheterologous proteins has been carried out. Examples of such hostorganisms include bacteria (e.g., E. coli), yeast (e.g., Saccharomycescerevisiae, Candida tropicalis, Hansenula polymorpha and P. pastoris;see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555 and4,855,231), mammalian cells (e.g., HEK293, CHO, CV-1, and Ltk cells),insect cells, and the like.

The present invention also provides a diagnostic system, preferably inkit form, for assaying for the presence of CRF-R protein, CRF-Rpolypeptide fragments or analogs, or CRF peptide in a fluid or tissuesample. A suitable diagnostic system includes, in an amount sufficientfor at least one assay, a CRF-R protein (or polypeptide fragmentthereof) and/or a subject antibody as a separately packagedimmunochemical reagent. Instructions for use of the packaged reagent arealso typically included.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

In one embodiment, a diagnostic system for assaying for the presence orquantity of CRF-R (or more likely a fragment of CRF-R) in a vascularfluid sample, such as blood, plasma, or serum, or in a tissue sample,comprises a package containing at least one CRF-R protein or polypeptidefragment thereof of this invention. In addition, a diagnostic systemcontaining at least one CRF-R (or polypeptide fragment thereof) can beused to detect the level of CRF peptide present in a vascular fluidsample or to detect the presence of an intracellular G-protein.

In another embodiment, a diagnostic system of the present invention forassaying for the presence or amount of CRF-R or fragment or analogthereof in a sample includes an anti-CRF-R antibody composition of thisinvention.

In yet another embodiment, a diagnostic system of the present inventionfor assaying for the presence or amount of CRF-R or a CRF-R polypeptidein a sample contains at least one CRF-R (or polypeptide fragmentthereof) and an anti-CRF-R antibody composition of this invention.

In preferred embodiments, a diagnostic system of the present inventionfurther includes a label or indicating means capable of signaling theformation of a complex containing a nucleic acid probe, protein,polypeptide, or antibody molecule of the present invention.

Also contemplated are immunohistochemistry diagnostic systems forcarrying out post-mortem diagnosis of mammalian tissue samples for thepresence of CRF-R, which employ the anti-CRF-R antibodies describedherein. For details on such diagnostic systems see, for example, Potteret al., PNAS, 89:4192-4296 (1992), incorporated herein by reference.

In yet another embodiment of the present invention, a hybridizationhistochemistry diagnostic system is provided. This diagnostic system isuseful for assaying for the presence or amount of nucleic acid encodingCRF-R (e.g., CDNA or mRNA) in a sample (e.g., vascular fluid or cellulartissue). This diagnostic system employs at least one CRF-R encodingnucleic acid probe of this invention.

The word “complex” as used herein refers to the product of a specificbinding reaction such as an antibody:antigen, receptor:ligand,protein:protein, or nucleic-acid-probe:nucleic-acid-target reaction.Exemplary complexes are immunoreaction products and CRF:CRF-R complexes.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal. Any label or indicating means can be linked to or incorporatedin a nucleic acid probe, an expressed protein, polypeptide fragment, orantibody molecule that is part of an antibody or monoclonal antibodycomposition of the present invention, or used separately. These atoms ormolecules can be used alone or in conjunction with additional reagents.Such labels are themselves well-known in clinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturation to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyanate (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB-200-SC), and the like. A description ofimmunofluorescence analytic techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In suchcases where the principal indicating group is an enzyme, additionalreagents are required for the production of a visible signal. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-azino-di-(3-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

In another embodiment, radioactive elements are employed as labelingagents. An exemplary radiolabeling agent is a radioactive element thatproduces gamma ray emissions. Elements which emit gamma rays, such as¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I and ⁵¹Cr, represent one class of radioactiveelement indicating groups. Particularly preferred is ¹²⁵I. Another groupof useful labeling means are those elements such as ¹¹C, ¹⁸F, ¹⁵O and¹³N which emit positrons. The positrons so emitted produce gamma raysupon encounters with electrons present in the animal's body. Also usefulis a beta emitter, such as ³²P, ¹¹¹indium or ³H.

The linking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptides, and proteins, is well known in theart. For instance, antibody molecules can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

The diagnostic systems can also include, preferably as a separatepackage, a specific binding agent. A “specific binding agent” is amolecular entity capable of selectively binding a reagent species of thepresent invention or a complex containing such a species, but is notitself a polypeptide or antibody molecule composition of the presentinvention. Exemplary specific binding agents are second antibodymolecules (e.g., anti-Ig antibodies), complement proteins or fragmentsthereof, S. aureus protein A, and the like. Preferably the specificbinding agent binds the reagent species when that species is present aspart of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits can be used in an “ELISA” format to detect thequantity of CRF, CRF-R, or CRF:CRF-R complex in a vascular fluid samplesuch as blood, serum, or plasma or in a mammalian tissue sample. “ELISA”refers to an enzyme-linked immunosorbent assay that employs an antibodyor antigen bound to a solid phase and an enzyme-antigen orenzyme-antibody conjugate to detect and quantify the amount of anantigen present in a sample. A description of the ELISA technique isfound in Chapter 22 of the 4th Edition of Basic and Clinical Immunologyby D. P. Sites et al., published by Lange Medical Publications of LosAltos, Calif. in 1982 and in U.S. Pat. No. 3,654,090, U.S. Pat. No.3,850,752; and U.S. Pat. No. 4,016,043, which are all incorporatedherein by reference.

Thus, in preferred embodiments, CRF-R protein, a CRF-R polypeptidefragment thereof, a polyclonal anti-CRF-R antibody, or a monoclonalanti-CRF-R antibody is affixed to a solid matrix to form a solid supportthat comprises a package in the subject diagnostic systems. A reagent istypically affixed to a solid matrix by adsorption from aqueous medium,although other modes of affixation applicable to proteins andpolypeptides well known to those skilled in the art can be used.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include cross-linked dextran (available fromPharmacia Fine Chemicals; Piscataway, N.J.); agarose; beads ofpolystyrene about 1 micron to about 5 millimeters in diameter (availablefrom Abbott Laboratories; North Chicago, Ill.); polyvinyl chloride;polystyrene; cross-linked polyacrylamide; nitrocellulose- or nylon-basedwebs such as sheets, strips or paddles; or tubes, plates or the wells ofa microtiter plate such as those made from polystyrene orpolyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials contemplated herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems. The term“package” refers to a solid matrix or material such as glass, plastic(e.g., polyethylene, polypropylene and polycarbonate), paper, foil, andthe like, capable of holding within fixed limits a diagnostic reagentsuch as a protein, polypeptide fragment, antibody or monoclonal antibodyof the present invention. Thus, for example, a package can be a bottle,vial, plastic or plastic-foil laminated envelope container, or the like,used to contain a diagnostic reagent. Alternatively, the container usedcan be a microtiter plate well to which microgram quantities of adiagnostic reagent have been operatively affixed, i.e., linked so as tobe capable of being immunologically bound by an antibody or polypeptideto be detected.

In normal individuals, the levels of CRF can vary from about 1 to 28picograms per milliliter of vascular fluid. However, during the lasttrimester of pregnancy, it has been found that there is a tendency forCRF levels to prematurely increase. It is believed that this increase isassociated with pregnancy-induced hypertension. Monitoring the change inthe level of CRF could facilitate the prediction of the possibility ofpremature labor, which can be avoided by appropriate treatment.

Thus, by monitoring the level of CRF, an abnormal increase indicative ofa potential pathological problem in pregnancy can be detected at anearly stage. Because normal hypertension is now believed to be eithercaused (or accompanied by) a higher CRF/“CRF-binding protein” ratio thannormal, monitoring the level of CRF facilitates the prediction ofparticular patients who are predisposed to such diseases, and permitstherapeutic intervention—as for example by administering dosages ofCRF-R protein or polypeptide fragments thereof. By the administration ofCRF-R or fragments thereof to treat such pregnancy related disorders,CRF levels can be returned to normal, thus facilitating the normalgrowth of the fetus.

The present invention contemplates various immunoassay methods fordetermining the amount of CRF-R in a biological fluid or tissue sampleusing a CRF-R, a polypeptide fragment thereof (including immunologicfragments, i.e., fragments capable of generating an immune response, asmore fully described in Harlowe and Lane, Antibodies: A LaboratoryManual, p. 76 (Cold Spring Harbor Laboratory, 1988)), an anti-CRF-Rpolyclonal or monoclonal antibody of this invention as an immunochemicalreagent to form an immunoreaction product whose amount relates, eitherdirectly or indirectly, to the amount of CRF-R in the sample. Alsocontemplated are immunoassay methods for determining the amount of CRFpeptide in a biological fluid sample using a CRF-R or a polypeptidefragment thereof as a reagent to form a product whose amount relates,either directly or indirectly, to the amount of CRF in the sample.

Various well-known heterogenous and homogenous protocols, eithercompetitive or noncompetitive, solution-phase or solid-phase, can beemployed in performing assay methods of the present invention. Thoseskilled in the art will understand that there are numerous well knownclinical diagnostic chemistry procedures in which an immunochemicalreagent of this invention can be used to form an immunoreaction productwhose amount relates to the amount of CRF-R or CRF present in a bodysample.

In one embodiment, the detection of CRF-R protein or polypeptidefragments in a body sample is utilized as a means to monitor the fate oftherapeutically administered CRF-R or polypeptide fragments according tothe therapeutic methods disclosed herein.

Also contemplated are immunological assays capable of detecting theformation of immunoreaction product formation without the use of alabel. Such methods employ a “detection means”, which means arethemselves well-known in clinical diagnostic chemistry. Exemplarydetection means include biosensing methods based on detecting changes inthe reflectivity of a surface, changes in the absorption of anevanescent wave by optical fibers, changes in the propagation of surfaceacoustical waves, and the like.

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallycompatible carrier together with a CRF-R protein, CRF-R polypeptidefragment, or anti-CRF-R antibody, as described herein, dissolved ordispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not immunogenic when administered to amammal or human patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically compatible” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset, and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well known in the art.Typically such compositions are prepared as injectables either as liquidsolutions or suspensions; however, solid forms suitable for solution, orsuspension, in liquid prior to use can also be prepared. The preparationcan also be emulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient inamounts suitable for use in the therapeutic methods described herein.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like, as well as combinations of any two or morethereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and the like, which enhance the effectiveness ofthe active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable nontoxic salts include the acid additionsalts (formed with the free amino groups of the polypeptide) that areformed with inorganic acids such as, for example, hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolicacid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid,naphthalene sulfonic acid, sulfanilic acid, and the like.

Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and the like; and organic bases such asmono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine,diisopropyl amine, methyl amine, dimethyl amine, and the like) andoptionally substituted ethanolamines (e.g., ethanolamine,diethanolamine, and the like).

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH, physiological saline orboth, such as phosphate-buffered saline. Still further, aqueous carrierscan contain more than one buffer salt, as well as salts such as sodiumand potassium chlorides, dextrose, polyethylene glycol and othersolutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary additional liquid phases includeglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

As previously indicated, administration of the CRF-Rs or polypeptidefragments thereof is effective to reduce vascular fluid CRF levels orhigh ACTH levels in mammals caused by excessive CRF, which is referredto herein as “CRF-induced ACTH release.” In this manner, the CRF-Rs areuseful in treating high cortisol (i.e., glucocorticoids) levels whichare associated with hypercortisolemia, Cushing's Disease, alcoholism,anorexia nervosa and similar diseases. Likewise, these CRF-Rs areconsidered to have utility in combatting pituitary tumors that produceCRF—particularly in maintaining stability in a patient until such atumor can be surgically removed.

In accordance with the present invention, CRF-RB₁ has surprisingly beenfound to be abundantly expressed in the heart. In the isolated perfusedheart, the addition of CRF into the left atrium induces a prolongeddilatory effect on coronary arteries, transiently produces a positiveionotropic effect, and stimulates the secretion of atrial natriureticpeptide (Saitoh et al., Gen. Pharmac. (England) 21:337-342 (1990); andGrunt et al., Horm. Metab. Res. (Germany) 24:56-59 (1992)). Thesurprising finding of CRF-RB₁ expression in the blood vessels of theheart raises the possibility that CRF (or other natural or pharmacologicligands for this receptor) might regulate cardiac perfusion.Furthermore, it is expected that other vascular beds, such as thesuperior mesenteric, known to be dilated by CRF and related ligands,will also be found to be regulated by CRF and receptors therefor.

Accordingly, since it is known that CRF has a number of biologicaleffects in the heart, it is contemplated that the CRF-R proteins,fragments thereof, or agonists/antagonists thereof (e.g., anti-CRF-Rantibodies), can be effectively used to selectively modulate the actionof CRF on the heart, particularly in methods to increase the level ofCRF that can act on the heart blood vessels to maintain patency.

In accordance with the present invention, the CRF-RB gene has also beenfound to be expressed in the gastrointestinal tract, as shown, forexample, by the presence of CRF-RB₁ mRNA in the submucosal and deeperregions of the duodenum. Accordingly, the CRF-RB₁ receptor may mediatesome of the direct stimulatory effects of CRF on the GI tract that havebeen described. For example, CRF acts on the gut in vitro to depolarizemyenteric neurons in the small intestine (Hanani and Wood, Eur. J.Pharmacol. (Netherlands) 211:23-27 (1992)). In vivo studies withintravenously administered CRF and CRF antagonists are consistent with adirect effect of CRF to control gastric emptying and intestinal motility(Williams et al., Am. J. Physiol. 253:G582-G586 (1987); Lenz, H. J.,Horm. Metab. Res. Suppl. 16:17-23 (1987); and Sheldon et al., Regul.Pept. 28:137-151 (1990)). In addition, CRF immunostaining is present atmany levels of the GI tract (Nieuwenhuyzen-Kruseman et al., Lancet2:1245-1246 (1982); Petrusz et al., Federation Proc. 44:229-235 (1985);and Kawahito et al., Gastroenterology 106:859-865 (1994)).

Thus, CRF-Rs (e.g., CRF-RB), fragments thereof, or agonists/antagoniststhereof (e.g., anti-CRF-R antibodies), are contemplated for use intreating gastrointestinal disorders, such as irritable bowel syndrome.In addition, CRF antagonists that are selective for CRF-RB are alsocontemplated for usef in therapeutic methods to treat irritable bowelsyndrome.

In addition, the presence of CRF-RB in the epididymis may enable localcommunication with spermatozoa, which are reported to possessimmunoreactive CRF (Yoon et a., Endocrinology 122:759-761 (1988)). Thus,CRF-Rs are also contemplated for use in treating fertility disorders.

The CRF-R proteins and fragments thereof are also useful to treatabnormalities, such as, for example, preeclampsia (toxemia ofpregnancy), which occur during pregnancy; for example, they can be usedto reduce pregnancy-induced complications and increased CRF levels whichcan otherwise result in excessive release of ACTH. In addition, CRF-Rproteins or fragments thereof can be administered to sequester CRF fromvascular fluid, thereby reducing the ratio of CRF/“CRF-binding protein”present in a patient, wherein it is beneficial to reduce the levels offree CRF (i.e., CRF not bound to CRF-BP) in the vascular fluid sample.CRF-binding protein (CRF-BP) is an extracellular serum protein describedin Potter et al., supra. The IV administration of CRF-Rs may also beemployed in certain instances to modulate blood pressure and therebycombat hypotension.

Since CRF is a known modulator of the immune system, it is contemplatedthat the administration of CRF-R protein or fragments thereof may beuseful to locally treat, i.e., by direct injection into the affectedjoint, arthritis and other similar ailments. CRF is known to have anumber of biological effects on the pituitary, and accordingly, theCRF-R proteins can be used to modulate the action of CRF on thepituitary. Furthermore, it is well known that CRF has a number ofbiological effects in the brain; therefore, it is contemplated that theCRF-R proteins can be effectively used to modulate the action of CRF onthe brain, particularly with respect to control of appetite,reproduction, growth, anxiety, depression, fever and metabolism, as wellas the regulation of blood pressure, heart rate, blood flow, and thelike.

Thus, the present invention provides for a method for modulating theaction of CRF in mammals comprising administering a therapeuticallyeffective amount of a physiologically acceptable composition containingCRF-R protein or polypeptide fragment of the present invention. Inaddition, the stimulation of ACTH release by CRF can be enhanced bytransfecting the subject with a tissue specific CRF-encoding construct.

In another embodiment, the present invention provides a method fortreating a pregnancy-related pathological disorder in mammals comprisingadministering a therapeutically effective amount of a physiologicallyacceptable composition containing a CRF-R protein or polypeptidefragment of the present invention, said amount being effective tosequester CRF, thereby producing a CRF/“CRF-binding protein” ratiowithin the normal range for a pregnant female.

Also, as earlier indicated, the administration of anti-CRF-R antibodiesdescribed herein is effective to modulate the biological effect ofCRF-Rs when administered in vivo. For example, an anti-CRF-R antibody ofthis invention can be used in the above-described mammalian therapeuticmethods to: neutralize or counteract the effect of CRF-R, increase thelevel of free CRF (e.g., CRF not bound by CRF-R), decrease CRF-inducedACTH release, or decrease the level of ACTH-induced glucocorticoids in asubject. Because increasing the level of free CRF increases the level ofCRF-induced ACTH release, which increases glucocorticoid production,these therapeutic methods are useful for treating certain physiologicalconditions where increasing the level of glucocorticoids in a patient'svascular fluid is therapeutically effective, such as conditions ofinflammation or Addison's Disease, and the like.

Administration of antibodies for this purpose would be carried out alongthe lines and in amounts generally known in this art, and moreparticularly along the lines indicated herein with respect toadministration of the protein itself.

As described herein, a therapeutically effective amount is apredetermined amount calculated to achieve the desired effect, e.g., todecrease the amount of CRF, ACTH, or decrease the CRF/“CRF-bindingprotein” ratio in a patient. The required dosage will vary with theparticular treatment and with the duration of desired treatment;however, it is anticipated that dosages between about 10 micrograms andabout 1 milligram per kilogram of body weight per day will be used fortherapeutic treatment. It may be particularly advantageous to administersuch compounds in depot or long-lasting form as discussed hereinafter. Atherapeutically effective amount is typically an amount of a CRF-Rprotein or polypeptide fragment thereof that, when administered in aphysiologically acceptable composition, is sufficient to achieve aplasma concentration of from about 0.1 mg/ml to about 100 mg/ml,preferably from about 1.0 mg/ml to about 50 mg/ml, more preferably atleast about 2 mg/ml and usually 5 to 10 mg/ml. Antibodies areadministered in proportionately appropriate amounts in accordance withknown practices in this art.

The level of ACTH present in a patient, particularly in the plasma, canbe readily determined by routine clinical analysis. In addition, changesin ACTH levels can be monitored during a treatment regimen to determinethe effectiveness of the administered CRF-R protein or polypeptidefragment over time.

Thus, the present therapeutic method provides an in vivo means fordecreasing ACTH levels in a subject displaying symptoms of elevatedserum ACTH, or is otherwise at medical risk by the presence of serumACTH, wherein it is beneficial to reduce the levels of ACTH. Inaddition, the present therapeutic method provides an in vivo means fordecreasing ACTH-induced cortisol levels (e.g., glucocorticoids) in ahuman patient displaying symptoms of elevated serum cortisol.

Likewise, the level of CRF present in a patient, particularly in theplasma, can be readily determined by the diagnostic methods and kitsprovided herein and readily manipulated by administering CRF-R, analogsthereof, or anti-CRF-R antibodies.

Thus, the present therapeutic method provides an in vivo means fordecreasing the CRF/CRF-BP ratio in a subject displaying symptoms ofelevated serum CRF/CRF-BP levels, or is otherwise at medical risk by thepresence of an elevated serum CRF/CRF-BP ratio, wherein it is beneficialto reduce the levels of free CRF (i.e., CRF not bound to CRF-BP) in thevascular fluid sample.

CRF-R protein(s) (or functional fragments thereof) should beadministered under the guidance of a physician. Pharmaceuticalcompositions will usually contain the protein in conjunction with aconventional, pharmaceutically-acceptable carrier. For treatment,substantially pure synthetic CRF-R or a nontoxic salt thereof, combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition, is preferably administered parenterally to mammals,including humans, either intravenously, subcutaneously, intramuscularly,percutaneously, e.g. intranasally, or intracerebroventricularly; oraladministration is possible with an appropriate carrier.

Therapeutic compositions containing CRF-R polypeptide(s) of thisinvention are preferably administered intravenously, as by injection ofa unit dose, for example. The term “unit dose,” when used in referenceto a therapeutic composition of the present invention, refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent, i.e., carrier, or vehicle.

Compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by repeated doses at one or more intervals by a subsequentinjection or other administration. Alternatively, continuous intravenousinfusion sufficient to maintain concentrations in the blood in theranges specified for in vivo therapies are contemplated.

As an aid to the administration of effective amounts of a CRF-Rpolypeptide, a diagnostic method of this invention for detecting a CRF-Rpolypeptide in the subject's blood is useful to characterize the fate ofthe administered therapeutic composition.

It may also be desirable to deliver CRF-R over prolonged periods oftime, for example, for periods of one week to one year from a singleadministration, and slow release, depot or implant dosage forms may beutilized. For example, a dosage form may contain a pharmaceuticallyacceptable non-toxic salt of the compound which has a low degree ofsolubility in body fluids, for example, an acid addition salt with thepolybasic acid; a salt with a polyvalent metal cation; or combination ofthe two salts. A relatively insoluble salt may also be formulated in agel, for example, an aluminum stearate gel. A suitable slow releasedepot formulation for injection may also contain CRF-R or a salt thereofdispersed or encapsulated in a slow degrading, non-toxic ornon-antigenic polymer such as a polylactic acid/polyglycolic acidpolymer, for example, as described in U.S. Pat. No. 3,773,919. Thesecompounds may also be formulated into silastic implants.

As additional examples of the utility of invention compositions, nucleicacids, receptors and/or antibodies of the invention can be used in suchareas as the diagnosis and/or treatment of CRF-dependent tumors,enhancing the survival of brain neurons, inducing abortion in livestockand other domesticated animals, inducing twinning in livestock and otherdomesticated animals, and so on.

In addition, invention cDNAs described herein encoding CRF-Rs (e.g.,CRF-RA and CRF-RB) can be used to isolate genomic clones encoding therespective CRF-R gene. The 5′ regulatory region of the isolated CRF-Rgene can be sequenced to identify tissue-specific transcription elements(i.e., promoter). The tissue-specific CRF-R promoters obtained areuseful to target various genes to cells that normally express CRF-Rs.For example, an adenovirus vector, having DNA encoding a cytotoxicprotein and a tissue-specific CRF-R promoter for pituitary corticotropiccells, can be used as means for killing pituitary corticotropic tumorcells.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., MolecularCloning: A Laboratory Manual (2 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methodsin Molecular Biology, Elsevier Science Publishing, Inc., New York, USA(1986); Methods in Enzymology: Guide to Molecular Cloning TechniquesVol.152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., SanDiego, USA (1987); or Harlowe and Lane, Antibodies: A Laboratory Manual,p. 76 (Cold Spring Harbor Laboratory, 1988).

Double-stranded DNA was sequenced by the dideoxy chain terminationmethod using the Sequenase reagents from US Biochemicals. Comparison ofDNA sequences to databases was performed using the FASTA program[Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988)].Tyr-ovine CRF used for iodination was purchased from Peninsula.

Example 1 Isolation of cDNA Encoding a Human CRF-R

A cDNA library of approximately 1.5×10⁶ independent clones from humanpituitary corticotrope adenoma (Cushing's Tumor) cells was constructedin the mammalian expression vector, pcDNA1, and screened using anexpression cloning approach [Gearing et al., EMBO J. 8, 3667-3676(1989)] based on the ability of single transfected cells to detectablybind labeled ¹²⁵I-Tyr-ovine CRF. Binding was assessed by performing thetransfections and binding reactions directly on chambered microscopeslides, then dipping the slides in photographic emulsion, developing theslides after 3-4 days exposure, and analyzing them under a microscope.The possibility of detecting expressed CRF Binding Protein, CRF-BP,rather than authentic CRF-R, was minimized by the selection of an ovineCRF related tracer known to have high affinity for the receptor but lowaffinity for CRF-BP. Cells which had been transfected with CRF receptorcDNA, and consequently bound radioactive CRF, were covered with silvergrains.

Polyadenylated RNA was prepared from human pituitary corticotropeadenoma cells. Corresponding cDNA was synthesized and ligated into theplasmid vector pcDNA1 using non-palindromic BstXI linkers and used totransform MC1061/P3 cells, yielding a library of approximately 1.5×10⁶primary recombinants. The unamplified cDNA library was plated atapproximately 5000 clones per 100 mm plate. The cells were then scrapedoff the plates, frozen in glycerol, and stored at −70° C.

Mini-prep DNA was prepared from each pool of 5,000 clones using thealkaline lysis method [Maniatis et al. Molecular Cloning (Cold SpringHarbor Laboratory (1982)]. Approximately 1/10 of the DNA from amini-prep (10 ml of 100 ml) was transfected into COSM6 cells, and thecells screened for the capacity to bind iodinated Tyr ovine CRF.

More specifically, 2×10⁵ COS cells were plated on chambered microscopeslides (1 chamber-Nunc) that had been coated with 20 mg/ml poly-D-lysineand allowed to attach for at least 3 hours in DMEM and 10Fetal CalfSerum (complete medium). Cells were subjected to DEAE-Dextran mediatedtransfection as follows. 1.5 ml of serum-free Dulbecco's ModifiedEagle's medium (DMEM) containing 100 mM chloroquine was added to thecells. DNA was precipitated in 200 ml DMEM/chloroquine containing 500mg/ml DEAE-Dextran, then added to the cells. The cells were incubated at37° C. for 4 hours, then the media was removed and the cells weretreated with 10% DMSO in HEPES buffered saline for 2 minutes. The 10%DMSO was removed, and fresh complete media was added and the cellsassayed for binding 2 days later.

Transfected cells prepared as described above were washed twice withHEPES buffered saline (HDB) containing 0.1% ovalbumin, then incubatedfor 90 minutes at 22° C. in 0.7 ml HDB, 0.1% ovalbumin containing 10⁶cpm ¹²⁵I-Tyr-ovine CRF (approximately 1 ng, 300 pM). The cells were thenwashed 3× with cold HDB, 0.1% ovalbumin, and 2× with cold HDB, thenfixed for 15 minutes at 22° C. in 2.5% glutaraldehyde/HDB and washed 2×with HDB. The chambers were then peeled off the slides, and the slidesdehydrated in 95% ethanol, dried under vacuum, dipped in NTB2photographic emulsion (Kodak) and exposed in the dark at 4° C. for 3-4days. Following development of the emulsion, the slides were dehydratedin 95% ethanol, stained with eosin and coverslipped with DPX mountant(Electron Microscopy Sciences). The slides were analyzed under darkfieldillumination using a Leitz microscope.

Successive subdivision of a positive pool generated a single clone thatdemonstrated high affinity CRF binding (K_(d)=3.3±0.45 nM) when presentin COSM6 cell membranes. The clone containing sequence encodingCRF-receptor is referred to herein as “hctCRFR” and has been depositedwith ATCC under accession number 75474, and the receptor encoded therebyis referred to herein as hCRF-RA₁.

A phage lZapII library was also synthesized from the same humanCushing's tumor cDNA described above using NotI/EcoRI adapters. A 1.2 kbPstI fragment in the CRF-R coding region of clone “hctCRFR” was used toscreen the lZapII library at high stringency using standard methods. Ofthree positive clones identified, two were sequenced and found tocontain full length CRF-R cDNA without introns. The clones are labeled“CRF-R1” (also referred to herein as hCRF-RA₁) and “CRF-R2” (alsoreferred to herein as hCRF-RA₂), portions of which are set forth in SEQID NO:1 and SEQ ID NO:3, respectively. Clone CRF-R₁ (i.e., hCRF-RA₁)contains a 2584 bp insert with a 1245 bp open reading frame encoding a415 amino acid CRF-R protein. Clone CRF-R2 (also referred to herein ashCRF-RA₂), is an alternatively spliced variant sequence of CRF-R1 (i.e.,hCRF-RA₁) that has the 29 amino acids set forth in SEQ ID NO:4 insertedbetween amino acids 145-146 of SEQ ID NO:2.

Example 2 Expression of CRF Receptor mRNA

Using well-known autoradiographic methods for binding labelled CRF tovarious frozen tissue sections, the native CRF receptor has beendetected and shown to vary dynamically in the pituitary and variousbrain regions in experimental animals and in human beings where it isaltered in pathologic conditions including Alzheimer's Disease andsevere melancholic depression. Furthermore, receptors have been detectedin the periphery in organs such as the adrenal, ovary, placenta,gastrointestinal tract and the red pulp, macrophage rich area of thespleen and in sites of inflammation presumably corresponding to theactions of CRF within those tissues.

A Northern-blot assay was conducted by size-fractionating poly(A)⁺-RNA(derived from rat brain, rat pituitary, rat heart, and mouse AtT20corticotropic cells) on a denaturing formaldehyde agarose gel andtransferring the RNA to nitrocellulose paper using standard methods. Thenitrocellulose paper blot was prehybridized for 15 minutes at 68° C. inQuikHybÔ hybridization solution (Stratagene, La Jolla, Calif.) and 100mg/ml salmon sperm DNA. Next, the blot was hybridized in the samesolution at 68° C. for 30 minutes to a “hctCRFR”-derived randomly primed(Amersham, Arlington Heights, Ill.) 1.3 Kb PstI cDNA fragment thatcontained the majority of the cDNA region of CRF-R1 (i.e., hCRF-RA₁).The blot was washed twice at 21° C. in 2×SSPE and 0.15% Sodium DodecylSulfate (SDS) for 15 minutes. Next, the blot was washed twice at 60° C.in 0.2×SSPE and 0.1% SDS for 30 minutes. An autoradiogram of thenitrocellulose paper blot was developed using standard methods.

The results of the Northern-blot assay revealed the presence of a 2.7 KbCRF-R mRNA transcript in rat brain, rat pituitary, and in mouse AtT20corticotropic cells. CRF-R mRNA was not detected in the heart tissuesample.

Example 3 Pharmacologic Characteristics of hctCRFR Transiently Expressedin COSM6 Cells

Approximately 10⁶ COSM6 cells were transfected with either hctCRFR orrGnRHR (rat gonadotropin releasing hormone receptor) by the DEAE-dextranmethod and grown in 150 mm tissue culture dishes. Two days aftertransfection, the cells were washed twice with 1 ml HDB and weredetached by incubation for 15 min at room temperature in 0.5 mM EDTA inHDB. After pelleting, the cells were washed twice with HDB, and thenhomogenized in 5% sucrose (16 ml/150 mm dish). The homogenate wascentrifuged at 600×g for 5 minutes, and the resulting supernatant wascentrifuged at 40,000×g for 20 minutes. The resulting pellet (containingcrude membranes) was resuspended at 1-4 mg/ml in 10% sucrose, and usedin a competitive radioreceptor assay to measure binding to the CRF-R asdescribed in Perrin et al., Endoc., 118:1171 (1986).

Membrane homogenates (10-24 mg) were incubated at room temperature for90 minutes with 100,000 cpm ¹²⁵I-(Nle²¹, Tyr³²)-ovine CRF (1 mg CRF wasiodinated by chloramine T oxidation to a specific activity of 2,000Ci/mmol; iodinated CRF was purified by HPLC) and increasingconcentrations of unlabeled rat/human (r/h) CRF. The iodinated CRF andunlabeled r/h CRF were both diluted in 20 mM HEPES, 0.1% BSA, 10%sucrose, 2 mM EGTA to a final, pH 7.5 in a final volume of 200 ml andcontaining MgSO₄ to a final concentration of 10 mM. The reaction wasterminated by filtration through GF/C (Whatman) filters, prewetted with1% BSA, 10 mM HEPES, pH 7.5. The filters were washed 4 times with 1 ml0.1% BSA, 50 mM Tris, pH 7.5. Filter-bound radioactivity, indicating thepresence of CRF-R: ¹²⁵I-(Nle²¹, Tyr³²)-ovine CRF complex, was determinedby g-scintillation counting.

The results from an assay for the displacement of ¹²⁵I-(Nle²¹,Tyr³²)-ovine CRF by unlabeled human/rat CRF (r/h CRF) are shown inFIG. 1. The results show that native r/h CRF is able to displace labeledovine CRF in a dose-dependent manner from cells transfected withhctCRFR, but not from cells transfected with rGnRHR. This indicates thatthe hctCRFR clone encodes a receptor that displays pharmacologicspecificity characteristic of a physiologically relevant CRF-receptor(i.e., CRF-RA₁).

Example 4 Assay of CRF-R Mediated Stimulation of Intracellular cAMPLevels

To determine the possible linkage of CRF-R to multiple signalingpathways, the ability of CRF-R to stimulate cAMP formation inCRF-R-expressing COSM6 cells was investigated. To ensure that changes incAMP levels were not influenced by alterations in cAMPphosphodiesterase, the phosphodiesterase inhibitor3-isobutyl-1-methylxanthine (IBMX) was added to the medium. COSM6 cellswere trypsinized 24 hrs following transfection with either ctCRFR orrGnRHR in 150 mm dishes and were replated in 24-well plates (Costar) andallowed to express the receptors for another 24 hrs in 10% FCS, DMEM.

On the day of the stimulation, the medium was changed to 0.1 FCS, DMEMat least 2 hrs before a 30 minute preincubation with 0.1 mM IBMX ormedium. Test ligands (i.e., r/h CRF, sauvagine, salmon calcitonin,Vasoactive intestinal peptide (VIP), growth hormone releasing factor(GRF) were added in 0.1% BSA, 0.1% FCS, DMEM, and stimulation wascarried out for 30 minutes at 37° C., 7.5% CO₂. The medium was removedand the cells were extracted overnight with 1 ml ice-cold 95% EtOH-0.1MHCl at −20° C. Cyclic AMP (cAMP) levels were determined in duplicatefrom triplicate wells by RIA kit (Biomedical Technologies, Stoughton,Mass.) following the manufacturer's protocol.

The results are shown in FIGS. 2A, 2B and 2C. FIGS. 2A and 2B show thatCOSM6 cells transfected with the cloned hctCRFR respond to CRF with anapproximately 10-20 fold increase in intracellular cAMP over basal cAMPlevels. Several unrelated peptides have no effect on cyclic AMP levelsin the receptor transfected cells. FIG. 2C shows that the CRFantagonist, a helical (9-41) CRF, blocks the induction of cyclic AMP byr/h CRF.

Example 5 Isolation of cDNA Encoding a Rat CRF-R

Adult Sprague-Dawley rat whole brain poly (A)+RNA was used for thesynthesis of a cDNA library. Double-stranded cDNA was ligated toEcoRI-NotI adaptors (Pharmacia/LKB) and cDNAs greater than 2 kilobasepair (kb) were ligated into the 1 ZAPII vector (Stratagene, La Jolla,Calif.). The library was amplified once and approximately 7×10⁵ cloneswere screened by hybridization with the 1.2 kb PstI fragment of CRF-R1(e.g., CRF-RA₁) using standard methods. One of the positive clonesidentified was sequenced and found to contain full length CRF-R cDNA.The positive clone was labeled rat brain CRF-R (rbCRF-RA) and containsan approximate 2500 base pair (bp) insert with a 1245 bp open readingframe encoding a 415 amino acid CRF-R protein. The cDNA and amino acidsequences corresponding to “rbCRF-RA” are set forth in SEQ ID NOs: 5 and6, respectively.

Example 6 Isolation of Genomic DNA Encoding a Mouse CRF-RB₁

Approximately 7×10⁶ clones of a mouse phage genomic library (obtainedfrom Stratagene, La Jolla, Calif.) were screened by hybridization with aprobe comprising nucleotides 204-1402 of rat CRF-RA (see SEQ ID NO:5)using standard methods. Thus, hybridization was carried out in 5×SSPE,5×Denhardt's solution, and 0.5% SDS for 16 hours at 60° C. The filterswere washed twice at room temperature with 2×SSC, 0.1% SDS, then washedtwice at 60° C. with 2×SSC, 0.1% SDS.

One of the positive clones identified was sequenced and found to containan open reading frame encoding a partial CRF-RB₁ sequence derived fromtransmembrane domains 3 through 4 of CRF-RB₁. The positive clone waslabeled mouse CRF-RB₁ (mCRF-RB₁) and contains two exons, interrupted byan intron of about 450 nucleotides. The two exons combine to produce a210 base pair (bp) open reading frame encoding a 70 amino acid portionof a novel CRF-RB₁ protein. The cDNA and amino acid sequencescorresponding to “mCRF-RB₁” are set forth in SEQ ID NOs: 7 and 8,respectively.

Upon further sequencing of clone mCRF-RB₁, a third exon was revealedcontaining an additional 78 base pairs (corresponding to nucleotides895-972 of SEQ ID NO:9) of an open reading frame encoding the CRF-RB₁protein.

Example 7 Isolation of cDNA Encoding a Mouse CRF-RB₁

In order to obtain the cDNA corresponding to the new mouse CRF-RB₁receptor, a mouse heart phage library was screened by hybridization.Approximately 1.2×10⁶ phage plaques of an amplified oligo-dt primedmouse heart cDNA library in the lambda zap II vector (Stratagene) werescreened by hybridization. A probe for the CRF-RB₁ cDNA was prepared byPCR using [a³²P]-dCTP and the following primers:

-   sense: 5′ CTGCATCACCACCATCTTCAACT 3′ (SEQ ID NO:11); and-   antisense: 5′ AGCCACTTGCGCAGGTGCTC 3′ (SEQ ID NO:12).    The template used in generating the probe was plasmid DNA    corresponding to one exon of CRF-RB₁ extending from amino acids 206    to 246 of SEQ ID NO:10. PCR amplification was carried out for 30    cycles (denatured at 94° C. for 1 minute, annealed at 55° C. for 2    minutes and extended at 72° C. for 3 minutes) to yield a 123 bp    product corresponding to nucleotides 693-815 of SEQ ID NO:9.

For hybridization screening, the plaques were lifted onto nylonmembranes, then denatured, neutralized and rinsed. The membranes wereprehyrbridized at 42° C. in 0.6M NaCl, 60 mM sodium citrate,4×Denhardt's, 40 mM sodium phosphate, pH 6.5, 170 mg/ml salmon spermDNA, 0.1% SDS, 20% formamide. The membranes were then hybridized at 42°C. in the same solution plus 50% dextran sulfate with approximately 10⁶cpm of labeled probe per ml of solution. After hybridization, thefilters were washed with 0.3M NaCl, 30 mM sodium citrate, 0.1% SDS onceat room temperature, twice at 42° C., and twice at 50° C.

A positive plaque was isolated and purified in the next round of plaquehybridization. Helper phage R408 (Biorad) was used for in vivo excisionof the lambda zap II clone. The cloned receptor was sequenced on bothstrands by the dideoxy chain-termination method using the Sequenase kit(United States Biochemical). The clone encoding mouse CRF-RB₁ containeda 2.2 kb insert that included a 1293 base pair (bp) open reading frameencoding a protein of 431 amino acids. The full-length CRF-RB₁ receptorcDNA was subcloned into the expression vector pcDNA1 (Invitrogen) usingthe EcoRI restriction enzyme to produce the plasmid pCRF-RB₁.

Alignments of the nucleotide and amino acid sequences were carried outusing the Jotun-Hein weighted method and the PAM250 residue weighttable, respectively. CRF-RB₁ has 70% homology at the nucleotide leveland 68% homology at the amino acid level, to CRF-RA₁. FIGS. 3A and 3Bpresent the comparison between the amino acid sequences of CRF-RB₁ andCRF-RA₁. The alignment was selected to maximize the regions ofsimilarity. There is a putative signal peptide and five putativeN-glycosylation sites in the N-terminal domain in CRF-RB₁, as there arein CRF-RA₁. When comparing CRF-RB₁ to CRF-RA₁, there is 79% similarityin the seven transmembrane domains (TMD) and 84% similarity in theintracellular loops and the intracellular tail. Yet there is only 60%identity in the extracellular loops (ECL), and 40% identity in theN-terminal domain, which has an additional sixteen amino acids.

All but one of the putative phosphorylation sites in CRF-RA₁ are foundin CRF-RB₁, the missing one being in the C-terminus. CRF-RB₁ also has anextra cysteine in a region of the N-terminus which may be within acleaved putative signal peptide, as well as an extra cysteine at thejunction of extracellular loop-1 and transmembrane domain-3, so that theresidue may fall within this transmembrane domain.

Example 8 Pharmacologic Characteristics of CRF-RB₁ Transiently Expressedin COSM6 Cells

Using the methods described in Example 3, a radioreceptor assay wasconducted. Approximately 10 mg of pCRF-RB₁ plasmid DNA was transfectedinto COSM6 cells using the DEAE dextran method. Two days later, thecells were detached and crude membrane fractions were prepared and usedto measure binding by competitive displacement of ¹²⁵I-(Nle21,Tyr32)-ovine CRF. In order to calculate a K_(d), the displacement datawere analyzed using the Ligand program of Munson and Rodbard (1980),Anal. Biochem., 107:220-239.

The cloned CRF-RB₁ binds CRF with high affinity as determined by thecompetitive displacement of bound radioligand. From six experiments, thedissociation constant was determined to be approximately K_(d)=1.3±0.2nM (see FIG. 4). The binding is specific because the peptides GRF andVIP do not displace the bound radioligand. In addition, the CRF-Rs havehigh affinity for sauvagine, urotensin, and potent CRF antagonists, suchas [DPhe¹², Nle^(21,38)]-hCRF(9-41).

Example 9 Assay of CRF-RB₁ Mediated Stimulation of Intracellular cAMPLevels

Using methods described in Example 4, the ability of CRF-RB₁ tostimulate cAMP formation in CRF-RB₁-expressing COSM6 cells wasinvestigated. The plasmid PCRF-RB₁ was transfected into COSM6 cells. Oneday later, the cells were trypsinized and replated in 10% FBS, DMEM into24 or 48 well COSTAR tissue culture wells, and allowed to grow another24 hours. The medium was changed to 0.1% FBS, DMEM at least two hoursbefore treatments. The cells were preincubated for 30 minutes with 0.1mM 3-isobutyl-1-methylxanthine and then exposed to various peptides for30 minutes at 37° C. Intracellular cAMP was measured in duplicate fromtriplicate wells using an RIA kit (Biomedical Technologies, Stoughton,Mass.).

The results are presented in FIG. 5. The results indicate that CRFstimulates the accumulation of intracellular cAMP when CRF-RB₁ istransiently transfected into COSM6 cells. The EC₅₀ occurs between 1-10nM, and the dose response is similar to that seen when the mouse analogof CRF-RA₁ is transfected. Urotensin and sauvagine, which are members ofthe CRF peptide family, are equipotent to CRF in stimulatingintracellular cAMP accumulation. The peptides GRF and VIP do notstimulate cAMP accumulation (see FIG. 5). The CRF signal transduced bythe cloned CRF-RB₁ receptor is inhibited in the presence of 1 mM CRFantagonist, (DPhe¹²,Nle^(21,38))hCRF(12-41).

Example 10 RNase Protection Assay to Determine Tissue Distribution ofCRF-RB₁

The coding region for mouse receptor corresponding to CRF-RA₁ was clonedby the well-known RT-PCR method using primers based on the publishedsequence, and RNA from mouse AtT-20 cells (ATCC No. CCl 89) as template.Plasmid DNAs encoding amino acids 26-106 of mouse CRF-RA₁ (SEQ ID NO:13)and amino acids 1 to 132 of mouse CRF-RB₁ (i.e., amino acids 1-132 ofSEQ ID NO:9) were linearized, and antisense riboprobes were synthesizedusing SP6 RNA polymerase and [a-³²P]UTP. An internal loading controlantisense riboprobe of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)was synthesized using T7 RNA polymerase.

RNase protection assays were performed by hybridizing 30 mg of total RNAfrom mouse heart and brain tissues to 5×10⁵ cmp of labeled riboprobe at65° C. for 18 hours. This was followed by RNase digestion (180 mg/mlRNase A and 350 U/ml RNase T1) at 23° C. for 60 minutes, after whichsamples were run on 5% polyacrylamide, 8 M urea gels.

RNase protection analysis was used to investigate the relativeexpression of CRF-RA₁ and CRF-RB₁ in mouse brain and heart. Whereas bothreceptors are detected in the brain and the heart, the heart appearsprimarily to express CRF-RB₁ (which has also been reported to expressCRF mRNA). There are multiple protected fragments seen in the heart.Furthermore, using a 5′ CRF-RB₁ probe, the major protected fragment inthe brain is smaller than in the heart, and smaller than that expectedto be generated from the cloned CRF-RB₁.

Example 11 In-Situ Hybridization Assay to Determine Tissue Distributionof CRF-RB₁

The tissue distribution of CRF-RB₁ mRNA was further characterized by insitu hybridization histochemistry using ³⁵S-labeled antisense cRNAprobes. Six week old male C57BL/6 mice were perfused transcardially with4% paraformaldehyde in 0.1M borate buffer,and regularly spaced series of20-30 mm thick frozen sections through brain, heart, duodenum andtestis/epididymis were taken as described (Simmons et al., J.Histotechnology 12:169-181 (1989)). Radiolabeled antisense and sense(control) cRNA copies were synthesized from a 1.0 kb BamH1 digest ofCRF-RB₁ cDNA, encompassing 80 bp of the 5′ untranslated region and 926bp of the CRF-RB₁ coding sequence, subcloned into pBluescript KS vector(Stratagene, La Jolla, Calif.). ³²S-UTP was used as the radioactiveisotope for probe synthesis, and in situ hybridization was performed aspreviously described (Simmons et al., supra; and Imaki et al., BrainRes. 496:35-44 (1989)). Probes were labeled to specific activities of1-3 ×10⁹ dpm/mg, and hybridization was carried out under high stringencyconditions (50% formamide with final washes in 0.2×SSC at 70° C.).

Sense-strand cRNAs labeled to similar specific activities failed to showany suggestion of positive localizations when applied to tissue sectionsadjoining those in which antisense probes revealed robust signals.Consistent with cloning and RNase protection assay data, CRF-RB₁transcripts were detected in the heart, where labeling appeared most.prominently over perivascular cells, as well as in the epicardium. Inthe male reproductive tract, CRF-RB₁ mRNA was localized principally instromal tissue of the epididymis, while labeling in testis was at ornear background levels. CRF-RB₁ mRNA signal over duodenum appeared as adense band of silver grains over the submucosal layer, and,additionally, over isolated non-epithelial cells at the base of thevilli.

In the brain, CRF-RB₁ mRNA displayed a rather restricted distribution,which contrasts in extent and topography with that for CRF-RA₁ mRNA inrat. In the septal region, for example, CRF-RB₁ mRNA is expressed incircumscribed aspects of the lateral septal nucleus, while the CRF-RA₁transcript is seen over the medial septal complex. Other major sites ofCRF-RB₁ mRNA expression in the forebrain include circumscribed aspectsof the olfactory bulb, preoptic region, hypothalamus, and amygdala.

In each of these areas, the pattern of CRF-RB₁ expression is seen to bedistinct from that of the CRF-RA₁ in rat brain. It appears unlikely thatmajor species differences in CRF-R distribution are at play, since themouse CRF-RB₁ probe employed here yielded similar patterns ofhybridization in mouse and rat brain.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the nucleic acid sequence (and the deduced aminoacid sequence) of a cDNA encoding a human-derived CRF receptor of thepresent invention (i.e., hCRF-RA₁).

Sequence ID No. 2 is the deduced amino acid sequence of thehuman-derived CRF receptor set forth in Sequence ID No. 1.

Sequence ID No. 3 is the nucleic acid sequence (and the deduced aminoacid sequence) of a splice variant cDNA insert encoding a 29 amino acidinsert portion of the human-derived CRF receptor of the presentinvention. The splice variant cDNA insert is located between nucleotides516-517 of Sequence ID No:1 (thereby producing CRF-RA₂).

Sequence ID No. 4 is the deduced amino acid sequence of thehuman-derived CRF receptor splice variant insert set forth in SequenceID No. 3. The splice variant amino acid insert is located between aminoacids 145-146 of SEQ ID NO:2.

Sequence ID No. 5 is the nucleic acid sequence (and the deduced aminoacid sequence) of a cDNA encoding region of a rat-derived CRF receptorof the present invention (i.e., rCRF-RA).

Sequence ID No. 6 is the deduced amino acid sequence of the rat-derivedCRF receptor set forth in Sequence ID No. 5.

Sequence ID No. 7 is the nucleic acid sequence (and the deduced aminoacid sequence) of two exons (less the intervening intron sequence) of apartial genomic clone encoding a mouse-derived CRF receptor of thepresent invention (i.e., mCRF-RB₁).

Sequence ID No. 8 is the deduced amino acid sequence of thehuman-derived CRF receptor set forth in Sequence ID No. 7.

Sequence ID No. 9 is the nucleic acid sequence (and the deduced aminoacid sequence) of a cDNA encoding a type-B mouse-derived CRF receptor ofthe present invention (i.e., mCRF-RB₁).

Sequence ID No. 10 is the deduced amino acid sequence of themouse-derived CRF-RB receptor set forth in Sequence ID No. 9.

Sequence ID No. 11 is the “sense” probe described in Example 7.

Sequence ID No. 12 is the “antisense” probe described in Example 7.

Sequence ID No. 13 is the amino acid sequence of the mouse-derivedCRF-RA₁ receptor.

Sequence ID No. 14 is the nucleotide sequence of a splice variant cDNAincluding an insert encoding a 29 amino acid portion of thehuman-derived CRF receptor of the present invention. The splice variantcDNA insert is located between nucleotides 516-517 of Sequence ID No:1(thereby producing CRF-RA₂).

Sequence ID No. 15 is the amino sequence of a splice variant CRF(CRF-RA₂) including a 29 amino acid portion of the human-derived CRFreceptor of the present invention. The splice variant amino acid insertis located between amino acids 145-146 of SEQ ID NO:2.

1. An isolated mammalian G protein-coupled corticotropin-releasingfactor (CRF) receptor protein, wherein said protein is encoded by DNAthat hybridizes to the complement of polynucleotide sequence set forthin SEQ ID NO:14 under moderately stringent conditions, comprisinghybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C., so asto allow identification of sequences having at least 70% nucleic acididentity with respect to SEQ ID NO:14; wherein said receptor proteinbinds CRF; and wherein said protein is at least about 70% pure (byweight of total proteins).
 2. The isolated protein according to claim 1having sufficient binding affinity for CRF such that concentrations ofless than or equal to 10 nanomolar CRF occupy greater than or equal to50% of the binding sites of said receptor protein.
 3. The isolatedprotein according to claim 1, wherein said protein is encoded by DNAhaving at least 80% nucleic acid identity with respect to SEQ ID NO:14.4. The isolated protein according to claim 1, wherein said protein isencoded by DNA having at least 90% nucleic acid identity with respect toSEQ ID NO:14.
 5. The isolated protein according to claim 1 having theamino acid sequence set forth in SEQ ID NO:15.
 6. The isolated proteinaccording to claim 1 having a radioactive labelling element attachedthereto.
 7. The isolated protein according to claim 1, wherein saidisolated protein is a recombinant protein.
 8. A composition comprisingan isolated protein according to claim
 1. 9. An antigenic fragment of anisolated mammalian G protein-coupled corti cotropin-releasing factor(CRF) receptor protein; wherein said protein is encoded by DNA thathybridizes to the complement of polynucleotide sequence set forth in SEQID NO:14 under moderately stringent conditions, comprising hybridizationin 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C.,followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C., so as to allowidentification of sequences having at least 70% nucleic acid identitywith respect to SEQ ID NO: 14; wherein said receptor protein binds CRF;and wherein said antigenic fragment is at least about 70% pure (byweight of total proteins).
 10. A substantially pure polypeptidecomprising at Least 15 contiguous amino acids of the amino acid sequenceset forth in SEQ ID NO:15; wherein said polypeptide is at least about70% pure (by weight of total proteins).
 11. The polypeptide according toclaim 10, wherein a residue selected from the group consisting oftyrosine, cysteine, lysine, glutamic acid and aspartic acid has beenattached by a peptide bond to the carboxyl terminus of said polypeptide.12. A diagnostic kit for assaying for the presence in biological fluidsof CRF-R protein, CRF-R protein analogs, and/or CRF-R fragments, saidkit comprising: (a) an isolated mammalian G protein-coupledcorticotropin-releasing factor (CRF) receptor protein according to claim1, and/or (b) one or more antibodies generated against said protein orimmunologic fragment thereof.